WO1993024506A1 - COMPOSES LEWISC ET LacNAc MODIFIES A EFFET IMMUNOSUPPRESSEUR ET INDUCTEUR DE TOLERANCE - Google Patents

COMPOSES LEWISC ET LacNAc MODIFIES A EFFET IMMUNOSUPPRESSEUR ET INDUCTEUR DE TOLERANCE Download PDF

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WO1993024506A1
WO1993024506A1 PCT/US1993/004995 US9304995W WO9324506A1 WO 1993024506 A1 WO1993024506 A1 WO 1993024506A1 US 9304995 W US9304995 W US 9304995W WO 9324506 A1 WO9324506 A1 WO 9324506A1
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compound
group
hydrogen
alkyl
carbon atoms
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PCT/US1993/004995
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Robert M. Ippolito
Wasimul Haque
Cong Jiang
H. Rizk Hanna
Andre P. Venot
Pandurang V. Nikrad
Mohammed A. Kashem
Richard H. Smith
Om P. Srivastava
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Alberta Research Council
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages

Definitions

  • the present invention is directed to
  • Lewis c -YR and LacNAc-YR analogues pharmaceutical compositions containing such analogues, methods for their preparation and methods for their use.
  • Carbohydrates and/or oligosaccharides are present on a variety of natural and pathological glycoconjugates 1 .
  • carbohydrates and oligosaccharides containing ⁇ ialyl and/or fucosyl residues 3 are present in a number of products which have been implicated in a wide range of biological phenomena based, in part, on the concept of recognition signals carried by the carbohydrate structures and by their binding to specific ligands.
  • sialylated and sialylated/fucosylated oligosaccharide glycosides have been proposed as mediators of cell adhesion in that they are ligands for selectins (or LEC-CAM's) 4,5,6 * 7 .
  • Sialylated, fucosylated, and sialylated and fucosylated oligosaccharide structures relating to blood group determinants having a type I or a type II core structure, including Lewis x , Lewis*, sialyl Lewis x and sialyl Lewis* have also been shown by Ippolito et al. 2,13 to possess in vivo immunomodulating and tolerogenic properties in mammals including anti- inflammatory immunomodulating properties.
  • oligosaccharide glycosides containing a sialyl group and/or a fucosyl group as well as related compounds are difficult to chemically synthesize in high yield.
  • synthesis of the ⁇ Neu5Ac(2 ⁇ 3)/3Gal disaccharide unit in compounds such as sialyl Lewis x and sialyl Lewis* with anomeric specificity for the ⁇ (2 ⁇ 3) is known to be difficult.
  • Known chemical methodologies include a multistep synthesis which first generates a blocked ⁇ Neu5Ac(2 ⁇ 3)Gal disaccharide having a suitable leaving group at the reducing sugar terminus of the galactose 8 ' 9 .
  • This disaccharide is then reacted with a suitably protected GlcNAc-OR saccharide glycoside and then a suitably protected L-fucose derivative which, after deprotection, provides for the sialyl Lewis glycoside [ ⁇ Neu5Ac(2 ⁇ 3)/3Gal(1 ⁇ 4) [ ⁇ Fuc(l ⁇ 3) ]-3GlcNAc-OR] or the sialyl Lewis* glycoside [i.e, ⁇ .Neu5Ac(2 ⁇ 3)jSGal(l ⁇ 3)-[o.Fuc(l ⁇ 4) ]-)3GlcNAc-OR] where R is an aglycon of at least one carbon atom.
  • sialylation and fucosylation can be conducted as part of an overall chemical/enzymatic synthesis 2,9,10 of compounds such as sialyl Lewis* and sialyl Lewis", such processes require compatible sialyltransferases and fucosyltransferase ⁇ which are not always readily available.
  • sialyltransferases and fucosyltransferase ⁇ which are not always readily available.
  • the /3Gal(l ⁇ 3/4)/3GlcNAc ⁇ (2 ⁇ 3)sialyltransferase disclosed in the art for sialylating the 3Gal(1 ⁇ 4) ⁇ GlcNAc backbone is currently recovered from rat livers 11 .
  • the present invention is directed, in part, to the discovery that modified Lewis c -YR and modified LacNAc-YR compounds having a sulfate, a phosphate or a carboxylate containing group at the 2, 3 and/or 6- positions of the galactose unit possess good immunosuppressive and tolerogenic properties.
  • the 3-sulfate Lewis 0 -YR compound possesses approximately equivalent immunosuppressive properties as compared to sialyl Lewis x -YR. This result is particularly surprising insofar as unmodified Lewis*-YR and sialyl Lewis*-YR possess inferior immunosuppressive properties as compared to the 3-sulfate of Lewis c -YR.
  • these modified LacNAc-YR and modified Lewis c -YR compounds do not require formation of an ⁇ (2-+3) sialyl residue on the galactose or a fucosyl residue at the 3- or 4-position of the GlcNAc residue in order to impart biological activity. Accordingly, the synthesis of biologically active molecules is simplified and the inclusion of a required sialyl group or a required fucosyl group is avoided. However, in this regard, the optional inclusion of a sialyl group at the 6-position of the galactose having a sulfate, phosphate or -CHR 18 COOH group at the 2- or 3-position of the galactose is contemplated herein.
  • the present invention is directed to compounds of Formula I or II:
  • R is selected from the group consisting of hydrogen, a saccharide-OR 19 , an oligosaccharide-OR 19 having from 2 to 7 oligosaccharide units, or an aglycon having at least 1 carbon atom where R 19 is hydrogen or an aglycon of at least one carbon atom;
  • Y is selected from the group consisting of oxygen, sulfur, and -NH-;
  • R u and R 15 are independently selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms, each R ⁇ is selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms; each R 12 is alkyl of from 1 to 4 carbon atoms, R 3 is selected from the group consisting of hydrogen, fluoro, sulfate and hydroxy;
  • X. is selected from the group consisting of hydrogen, sialyl, sulfate, phosphate, and -CHR 18 C00H where R 18 is selected from the group consisting of hydrogen, alkyl of from 1 to 7 carbon atoms and -COOH;
  • X 2 is selected from the group consisting of hydrogen, sulfate, phosphate, and -CHR 18 COOH where R 18 is selected from the group consisting of hydrogen, alkyl of from 1 to 7 carbon atoms and -COOH; and pharmaceutically acceptable salts thereof; and with the proviso that either at least one of X- or X 2 is sulfate, phosphate or -CHR 18 COOH or R 3 is sulfate.
  • the compounds of Formula I and II are useful in modulating a cell-mediated immune inflammatory response and in particular a cell-mediated immune inflammatory response to an antigen.
  • the compounds of this invention are particularly useful in reducing antigen induced inflammation in a sensitized mammal.
  • the compounds of Formula I and II when administered to a sensitized mammal in response to an antigen challenge, such administration induces tolerance to later challenges from this same antigen.
  • administration of a compound of Formula I or II above is after initiation of the mammal's immune response but at or prior to one-half of the period of time required for the mammal to reach maximal inflammation.
  • the present invention is directed to a pharmaceutical composition suitable for administration to a mammal (e.g. , human) which comprises a pharmaceutically inert carrier and an effective amount of the compound of Formula I or Formula II to modulate a cell-mediated immune inflammatory responses in said mammal.
  • a mammal e.g. , human
  • the present invention is directed to a method for modulating a cell-mediated immune inflammatory response in a mammal which method comprises administering to said mammal an amount of a compound of Formula I or Formula II effective in modulating said immune response.
  • Figure 1 illustrates reaction schemes for the synthesis of partially blocked N-acetyl glucosamine derivatives which are then used to prepare either modified Lewis c -OR compounds or modified LacNAc compounds.
  • Figure 2 illustrates reaction schemes for the synthesis of partially blocked galactose derivatives which are then used to prepare either modified Lewis c - OR compounds or modified LacNAc-OR compounds.
  • Figure 3A illustrates one method for the preparation of 3-sulfated Lewis c -OR compounds.
  • Figure 3B illustrates one method for the preparation of 6-sulfated Lewis c -OR compounds.
  • Figure 4 illustrates methods for preparing differentially blocked /3Gal(l ⁇ 3)/3GlcNAc-OR compounds (Lewis c -OR) and derivatives thereof and methods for preparing differentially blocked SGal(l ⁇ 4) ->GlcNAc-OR (LacNAc-OR) compounds.
  • Figure 5 illustrates the synthesis of the 6-azido derivative of GlcNAc-OR.
  • Figure 6 illustrates the synthesis of the 6- alkoxy derivatives and the 6-deoxy derivatives of GlcNAc-OR.
  • Figure 7 illustrates the preparation of 3- hydroxy or 4-hydroxy blocked GlcNH 2 -OR where the amino group is protected as an N-phthalimido group.
  • the present invention is directed, in part, to the discovery of novel Lewis c -YR and novel LacNAc-YR analogues which, in mammals, including humans, are useful for in vivo modulation (e.g. , suppression) of a cell mediated immune response including cell-mediated and immune directed inflammatory responses to an antigen in a sensitized mammal (e.g., a DTH response).
  • novel Lewis c -YR and novel LacNAc-YR analogues which, in mammals, including humans, are useful for in vivo modulation (e.g. , suppression) of a cell mediated immune response including cell-mediated and immune directed inflammatory responses to an antigen in a sensitized mammal (e.g., a DTH response).
  • cell-mediated immune response in a mammal refers to those mammalian immune responses which are mediated by cell-cell interactions. Included within this term are cell mediated inflammatory responses to an antigen such as DTH responses as well as cell-mediated inflammatory responses arising from injury such as frost-bite injury, reperfusion injury, adult respiratory distress syndrome, and the like. Preferably, the cell-mediated immune response is a leucocyte-mediated response.
  • antigen refers to any protein, peptide, carbohydrate, nucleic acid or other non- endogenous substance which when exposed to a mammal induces an immune response in that mammal.
  • Disease conditions believed to be caused by antigen exposure include, by way of example, psoriasis, asthma, dermatitis, rheumatoid arthritis, delayed type hypersensitivity, inflammatory bowel disease, multiple scelorsis, viral pneumonia, bacterial pneumonia, and the like.
  • sensitized mammal refers to those mammals which have been previously exposed to an antigen and, accordingly, their immune systems have become educated to that antigen. Typically, initial exposure of an antigen to a mammal primes or educates the mammal's immune response to later exposure to that antigen with minimal inflammation during such initial exposure.
  • second immune response refers to the effector phase of a mammal's immune response to an antigen to which it has been previously been sensitized.
  • a mammal's secondary immune response is typically accompanied by inflammation at the point of antigen exposure.
  • periodic for maximal inflammation refers to the period of time typically required to achieve maximal inflammation in a sensitized mammal due to exposure to a specific antigen or to the period of time typically required to achieve maximal inflammation in a mammal due to an injury which induces a cell- mediated inflammatory response (e.g., myocardial infarction) . This period of time depends on several factors. For example, for inflammation due to injury, the period for maximal inflammation depends on factors such as the type and extent of injury.
  • this period is dependent on factors such as the specific antigen to which the mammal has been exposed, the particular mammalian species exposed to the antigen, etc. Accordingly, the period of time required to effect maximal antigen induced inflammation in-a sensitized mammal will vary for, by way of example, asthma as opposed to rheumatoid arthritis.
  • LacNAc refers to the disaccharide jSGal(l ⁇ 4)3GlcNAc Because of its relationship to the core structure of type II blood group determinants, the ⁇ Gal(1 ⁇ 4) ⁇ GlcNAc structure of LacNAc is often referred to as a type II structure.
  • LacNH 2 refers to the LacNAc derivative wherein the N-acetyl group of LacNAc has been replaced with an amine (-NH 2 ) .
  • LacN 3 refers to the LacNAc derivative wherein the N-acetyl group of LacNAc has been replaced with an azido (-N 3 ) .
  • Lewis 0 (sometimes referred to "Le°") refers to the disaccharide ⁇ Gal(l ⁇ 3)/3GlcNAc. Because of its relationship to the core structure of type I blood group determinants, the 3Gal(l ⁇ 3)?GlcNAc structure of Lewis 0 is often referred as a "type I structure”.
  • Lewis 0 modified in one or both of the galactose and N-acetylglucosamine saccharide units of Lewis 0 and which have an -YR substituent as defined above.
  • R substituent is an aglycon group
  • this group has at least one carbon atom, but nevertheless is different from glycoconjugates because such aglycon moieties are neither a protein nor a lipid capable of forming a micelle or other large aggregate structure.
  • modified LacNAc glycosides and derivatives thereof refer to derivatives of the LacNAc modified in one or both of the galactose and N-acetylglucosamine saccharide units of LacNAc and which have an -YR substituent as defined above.
  • R substituent is an aglycon group
  • this group has at least one carbon atom, but nevertheless is different from glycoconjugates because such aglycon moieties are neither a protein nor a lipid capable of forming a micelle or other large aggregate structure.
  • the aglycon moiety, R is selected from the group consisting of -(A)-Z wherein A represents a bond, an alkylene group of from 2 to 10 carbon atoms, and a moiety of the formula -(CH 2 -CR 20 G) n - wherein n is an integer equal to 1 to 5;
  • R 20 is selected from the group consisting of hydrogen, methyl, or ethyl;
  • G is selected from the group-consisting of hydrogen, halogen, oxygen, sulphur, nitrogen, phenyl and phenyl substituted with 1 to 3 substituents selected from the group consisting of amine, hydroxyl, ' halo, alkyl of from 1 to 4 carbon atoms and alkoxy of from 1 to 4 carbon atoms;
  • Z is selected from the group consisting of hydrogen, methyl, phen
  • aglycons are known in the art.
  • Ekborg, et al. 18 At the appropriate time during synthesis, the nitro group is reduced to an amino group which can be protected as N-trifluoroacetamido.
  • the trifluoro- acetamido group can later be removed thereby unmasking the amino group which can then be used to further functionalize the aglycon group.
  • aglycon containing sulfur is disclosed by Dahmen, et al. 19 .
  • the aglycon is derived from a 2-bromoethyl group which, in a substitution reaction with thionucleophiles, has been shown to lead to aglycons possessing a variety of terminal functional groups such as -OCH 2 CH 2 SCH 2 C0 2 CH 3 and -OCH 2 CH 2 SC 6 H A -pNH 2 .
  • Rana, et al. 20 discloses a 6-trifluoroacet- amidohexyl aglycon (-0-(CH 2 ) 6 -NHCOCF 3 ) in which the trifluoroacetamido protecting group can be removed unmasking the primary amino group which can then be used to further functionalize the aglycon group.
  • allyl aglycons can be derivatized in the presence of 2-aminoethane- thiol 25 to provide for aglycons -OCH 2 CH 2 CH 2 SCH 2 CH 2 NH 2 . Still other aglycons are illustrated hereinbelow.
  • the R group can be an additional saccharide-OR 19 or an oligosaccharide-OR 19 containing an aglycon at the reducing sugar terminus.
  • oligosaccharide refers to a carbohydrate structure having from 2 to about 7 saccharide units.
  • the particular saccharide units employed are not critical and include, by way of example, all natural and synthetic derivatives of glucose, galactose, N-acetylglucosamine, N-acetyl- galactosamine, fucose, sialic acid, 3-deoxy-D,L- octulosonic acid, and the like.
  • all saccharide units described herein are in their D form except for fucose which is in its L form.
  • sialic acid or "sialyl” means all naturally occurring structures of sialic acid and analogues of sialic acid.
  • Naturally occurring structures of sialic acid include, by way of example, 5-acetamido-3,5-dideoxy-D-glycero-D-galactononulo- pyranosylonic acid (“Neu5Ac”) , N-glycoyl neuraminic acid (Neu5Gc) and 9-O-acetyl neuraminic acid (Neu5,9Ac 2 ) .
  • Analogues of sialic acid refers to analogues of naturally occurring structures of sialic acid including those wherein the sialic acid unit has been chemically modified so as to introduce and/or remove one or more functionalities from such structures. For example, such modification can result in the removal of an -OH functionality, the introduction of an amine functionality, the introduction of a halo functionality, and the like.
  • analogues of sialic acid include, by way of example, 9-azido-Neu5Ac, 9-amino-Neu5Ac, 9-deoxy-Neu5Ac, 9-fluoro-Neu5Ac, 9- bromo-Neu5Ac, 7-deoxy-Neu5Ac, 7-epi-Neu5Ac, 7,8-bis- epi-Neu5Ac, 4-0-methyl-Neu5Ac, 4-N-acetyl-Neu5Ac, 4,7- di-deoxy-Neu5Ac, 4-oxo-Neu5Ac, as well as the 6-thio analogues of Neu5Ac
  • the nomenclature employed herein in describing analogues of sialic acid is as set forth by Reuter et al. 12
  • pharmaceutically acceptable salts includes the pharmaceutically acceptable addition salts of the compounds of Formula I or Formula II derived from a variety of organic and inorganic counter salts well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and .the like.
  • sulfate such as used to define the substituents -X 1 and -X 2 refers to substituents which, with the oxygen of a hydroxyl group of the galactose unit, form a sulfate group (i.e., -O-S(0) 2 -OH) .
  • X 1 or X 2 is a sulfate
  • the resulting -0X 1 and/or -0X 2 group is -0-S(0) 2 -0H, which readily forms pharmaceutically acceptable salts thereof (e.g., -0-S(0) 2 -0 " Na + )
  • the term “sulfate” as it is used for R 3 refers to the -0-S(0 2 )-0H group, which readily forms pharmaceutically acceptable salts thereof.
  • phosphate such as used to define the substituents -X. and -X 2 refers to substituents which, with the oxygen of a hydroxyl group of the galactose unit, form a phosphate group (i.e., -O-P(O)-(OH) 2 .
  • a phosphate group i.e., -O-P(O)-(OH) 2
  • X, or X 2 is a phosphate
  • the resulting -OX, and/or -OX 2 group is -O-P(O)-(OH) 2 , which readily forms pharmaceutically acceptable salts thereof (e.g., -0-P(0)-(0 " Na + ) 2 .
  • blocking group refers to any group which when bound to one or more hydroxyl groups of the galactose and/or the N-acetylglucosamine of the Lewis°-YR and LacNAc-YR compounds prevents reactions from occurring at these hydroxyl groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl group.
  • removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as benzyl, benzoyl, acetyl, chloroacetyl, benzylidine, t-butyldiphenyl- silyl and any other group that can be introduced either enzymatically or chemically onto a hydroxyl functionality and later selectively removed either by enzymatic or chemical methods in mild conditions compatible with the nature of the product.
  • One such contemplated blocking group is a ⁇ -galactose which can be removed enzymatically with an ⁇ -galactosidase.
  • Chemical modifications include introduction of the sulphate or phosphate group or a -OCHR 18 COOH at the 3 and/or 6 position of the terminal galactose; the introduction of a sulfate at the 2-position of the galactose; and optionally, the introduction of modification at the 2- and 6- positions of the
  • N-acetylglucosamine unit and/or introduction of deoxy or fluoro functionality at the 2-position of the galactose, and the like are N-acetylglucosamine units and/or introduction of deoxy or fluoro functionality at the 2-position of the galactose, and the like.
  • Lewis°-YR and LacNAc-YR and some analogues thereof are known in the art. These materials are generally assembled using suitably protected individual monosaccharide intermediates. The modifications to the final structures are accomplished using known methods for sulfation or phosphorylation after appropriate selective deblocking of the to-be functionalized hydroxyl group(s) of the fully blocked Lewis°-YR or LacNAc-YR.
  • the chemical synthesis of all or part of the these disaccharides first involves formation of a glycosidic linkage on the anomeric carbon atom of the reducing sugar.
  • an appropriately protected form of a naturally occurring or of a chemically modified saccharide structure (the glycosyl donor) is selectively modified at the anomeric center of the reducing unit so as to introduce a leaving group comprising halides, trichloroacetimidate, acetyl, thioglycoside, etc.
  • the donor is then reacted under catalytic conditions well known in the art with an aglycon or an appropriate form of a carbohydrate acceptor which possess one free hydroxyl group at the position where the glycosidic linkage is to be established.
  • aglycon moieties are known in the art and can be attached with the proper configuration to the anomeric center of the reducing unit.
  • Appropriate use of compatible blocking groups, well known in the art of carbohydrate synthesis, will allow selective modification of the synthesized structures or the further attachment of additional sugar units or sugar blocks to the acceptor structures.
  • the saccharide glycoside can be used to effect coupling of the galactose unit or chemically modified at selected positions.
  • chemical coupling of a naturally occurring or chemically modified saccharide unit to the saccharide glycoside is accomplished by employing established chemistry well documented in the literature. See, for example, Okamoto et al. 31 ,
  • Figure 1 illustrates the synthesis of numerous blocked derivatives of glucosamine and N-acetylglucosamine which are then useful in the preparation of blocked LacNH 2 -OR [jSGal(1 ⁇ 4)/3GlcNH 2 -OR] , LacNAc-OR [ ⁇ Gal(1 ⁇ 4) ⁇ GlcNAc-OR] , Lewis°-OR [3Gal(1-3)3GlcNAc-OR] , Lewis°-NH 2 -OR [/3Gal(l ⁇ 3) ?GlcNH 2 -OR] , etc. structures.
  • glucosamine hydrochloride is slurried in dichloroethane containing an equivalent of anhydrous sodium acetate to which acetic anhydride is added dropwise and, after addition is completed, the solution is refluxed for a period of from about 12-16 hours to provide for the peracylated compound 10 (about 3:1 ratio of a/ ⁇ ) .
  • the glucosamine hydrochloride is first taken up in methanol and then treated with 1 equivalent of metallic sodium to neutralize the HCl. Phthalic anhydride is then added quickly to the reaction mixture followed shortly thereafter by triethylamine to provide for the phthalimido derivative. This compound is then isolated and acetylated with acetic anhydride/pyridine using conventional techniques to provide for peracylated compound 1 having a phthalimide blocking group protecting the amine.
  • the aglycon is formed by conventional techniques.
  • compound 10 is converted to 1- ⁇ -chloro compound 2 by well known chemistry which involves bubbling saturating amounts of hydrogen chloride directly into a dichloroethane solution of compound 10.
  • the solution used to prepare compound 10 can be used in this reaction after that solution has been quenched into water to remove acetic anhydride and sodium acetate, dried and recovered.
  • the reaction generally proceeds over a period of about -4-6 days and hydrogen chloride is bubbled into the solution periodically (e.g., about once ever 1-2 days) .
  • the solution is quenched in aqueous sodium bicarbonate at about 0-5°C and the product is recovered after drying the organic layer and stripping the solution to provide for compound 2 (one spot on t.l.c)
  • Compound 2 is then converted to the l-3-(CH 2 ) 8 COOCH 3 aglycon by well known chemistry which involves reaction of compound 2 with HO(CH 2 ) 8 COOCH 3 in anhydrous dichloromethane containing molecular sieves in the presence of an equivalent amount of mercuric cyanide.
  • the reaction is generally conducted at room temperature for a period of about 12 to 24 hours.
  • reaction completion (as evidenced by t.l.c)
  • the reaction solution is filter through silica and the resulting solution is quenched by adding the reaction solution to cold water.
  • compound 5 can be blocked at the 3-hydroxyl group by reaction with, for example, allyl bromide and base (e.g., barium hydroxide/barium oxide) to provide for compound 8.
  • allyl bromide and base e.g., barium hydroxide/barium oxide
  • compound 5 contains a free hydroxyl group only at the 3-position of the blocked GlcNAc-OR saccharide, subsequent reaction with an appropriately blocked galactose will result in formation of a blocked type I structure [/3Gal(l ⁇ 3)/3GlcNAc-OR] .
  • compound 1 can be converted to compound 11 by reaction of compound 1 with an equivalent of p-chlorothiophenol in dichloromethane at room temperature in the presence of 2 equivalents of boron trifluoride etherate (BF 3 -etherate) to provide for compound 11.
  • boron trifluoride etherate BF 3 -etherate
  • compound 1 is converted to compound 12 (or the bromo analogue) by following similar procedures set forth above for compound 2.
  • Compound 13 is then converted to compound 14 with sodium methoxide/methanol and is then converted to compound 15 by reaction with bis[tributyltin] oxide in refluxing toluene containing tetraethylammonium bromide followed by reaction with benzyl bromide.
  • Compound 13 is then converted to compound 14 with sodium methoxide/methanol and is then converted to compound 15 by reaction with bis[tributyltin] oxide in refluxing toluene containing tetraethylammonium bromide followed by reaction with benzyl bromide.
  • compound 15 contains free hydroxyl groups at the 3- and 4-positions of the blocked GlcNAc- OR saccharide, subsequent reaction with an appropriate- ly blocked galactose will result in formation of both a type I structure [,3Gal(l ⁇ 3)3GlcNAc-OR] and type II structure [3Gal(l ⁇ 4)/3GlcNAc-OR] which are readily separated by conventional techniques including chromatography.
  • Compound 16 is prepared by treating p-chloro- thiophenol with 0.95 equivalents of potassium hydroxide in ethanol followed by heating the solution to about 40-50"C and then adding about 0.5 equivalents of compound 2 to the reaction solution. The reaction is maintained at 40-50°C for about 1-2 hours and the product 16 precipitates upon cooling the solution and is recovered by filtration.
  • D-galactose pentaacetate 26 is produced by slurring D-galactose and about an equimolar amount (e.g., about 1.1 equivalents) of sodium acetate (NaOAc) in dichloroethane (DCE) , heating to reflux and adding at least 5 equivalents of acetic anhydride (AcOAc) dropwise to the refluxing solution (about 80-85°C) and then maintaining the reaction system at this temperature for a sufficient period of time (about 16-32 hours) to result in formation of compound 26.
  • This procedure optimizes the yield of ⁇ -D-galactose pentaacetate 26 and controls the exotherm of heretofore known procedures.
  • the product is treated with approximately equimolar amounts of benzyl mercaptan (Ph-CH 2 -SH) and from about 1-3 (preferably two) equivalent of boron trifluoride etherate (BF 3 -OEt 2 ) in dichloromethane.
  • the reaction conditions are not critical and the reaction is preferably conducted at from about 0°C to about 30°C for a period abouut 6 to 16 hours to yield after crystallization from hot methanol or hot isopropanol 55-65% of benzyl 2,3,4,6- tetra-O-acetyl ⁇ -D-thiogalactopryanoside, compound 27.
  • Deacetylation under Zemplen conditions leads to compound 28.
  • Deacetylation reaction conditions are not critical and the reaction is generally conducted at room temperature for a period of from about 2 to about 15 hours. After the deacetylation reaction is complete (as judged by t.l.c), the solution is neutralized with an acid ion exchange resin, filtered and evaporated to dryness to provide for compound 28.
  • the residue is crystallized from hot acetone and the product is taken up in dimethylformamide or aoetonitrile and treated with from 1 to 2 equivalents (preferably 1.4 equivalents) of benzaldehyde dimethyl acetal and about 0.25 to 3 weight percent of p-toluenesulphonic acid (based on compound 28) .
  • the reaction conditions are not critical and preferably the reaction is conducted at room temperature and is generally complete in about 12 to 24 hours.
  • the benzyl 4,6-0- benzylidene ⁇ -D-thiogalactopyranoside, 29, is isolated and crystallized from hot isopropanol.
  • Benzyl 4,6-0-benzylidene-3-0-chloroacetyl- ⁇ -D-thiogalactopyranoside 30 is prepared by chloroacetylation using from about 1 to 3 (preferably 2) equivalents of chloroacetylchloride which is added to a dimethylformamide (DMF) solution containing benzyl 4,6-0-benzylidene ⁇ -D-thiogalacto-pyranoside 29.
  • the chloroacetylchloride is added dropwise while maintaining the DMF solution at from about -40° to about -15°C (preferably at -25°C) . Under these conditions, it is unexpectedly been found that the use of DMF permits selective chloroacetylation of compound 29 without the need for additional base.
  • the reaction is generally complete in about 10-24 hours.
  • Benzyl 4,6-0-benzylidene-3-0-chloroacetyl- ⁇ - D-thiogalactopyranoside (compound 30) is benzoylated with at least 1 equivalent (and preferably about 2 equivalents) of benzoyl chloride in a suitable solvent containing a base (e.g., pyridine/methylene chloride) with from about 0.1 to about 1 weight percent of dimethylaminopyridine [DMAP] as a catalyst.
  • a base e.g., pyridine/methylene chloride
  • reaction conditions are not critical and preferably the reaction is conducted at from about 0*C to about 30°C and for about 1 to about 4 hours (preferably room temperature for 2 hours, to give crystalline benzyl 4,6-0-benzylidene 2-0-benzoyl-3-0-chloroacetyl- ⁇ -D- thio-galactopyranoside, compound 31, in approximately 10-20% overall yield from galactose.
  • the sulfates and phosphates of the galactose moiety of blocked Lewis°-YR and LacNAc-YR can also be made using compound 32 in the synthesis of these compounds.
  • This compound is made by direct benzoylation of both the 2,3-hydroxyl groups of compound 29.
  • both the 2 and 3 hydroxyl groups of galactose are then available for sulfation and phosphorylation and the selectivity is not as efficient.
  • Selectivity can be improved by, for example, conducting the sulfation reaction at a low temperature (e.g., -50°C) .
  • Compound 29 can be converted to the 2,3- dibenzoyl protected compound 32 in a manner similar to that described above for the preparation of compound 31. In this case, 3-5 equivalents of benzoyl chloride are generally employed.
  • Compounds 31 and 32 are converted to compounds 3: and 32a (shown in Figure 4) via known methodology (Norberg et al. 26 ) using bromine tetraethylammonium bromide.
  • compound 31 can be converted to compound 34 by contacting compound 31 with 80% acetic acid/water at approximately 50°C for about 1-2 ho rs. Compound 34 is then converted to compound 35 by treatment with acetic anhydride/pyridine in dichloromethane.
  • compound 32 is treated with sodium cyanoborohydride and eerie chloride to provide for the benzyl-2,3-0-dibenzoyl-4-0-benzyl-/3-D- thiogalactopyranoside (not shown) .
  • this compound is chloroacetylated at the 6-hydroxyl group.
  • the chloroacetyl group can be selectively removed (as described above) and then either phosphorylated or sulphated so as to provide for the 6- phosphate or 6-sulfate derivative.
  • Figure 3A illustrates one method for the preparation of 3-sulfated Lewis°-OR compounds.
  • compound 101 is prepared by reacting N-acetylglucosamine-OR (e.g., compound 4 in Figure 1) with C 6 H 5 CH(OCH 3 ) 2 in an acidic aoetonitrile or dimethylformamide (DMF) medium at from about 0° to about 50°C over 12-48 hours to provide for the 4,6-diprotected benzylidine compound 101.
  • N-acetylglucosamine-OR e.g., compound 4 in Figure 1
  • C 6 H 5 CH(OCH 3 ) 2 e.g., compound 4 in Figure 1
  • DMF dimethylformamide
  • N-iodosuccinimide is combined with about 1 equivalent of trifluoromethane- sulfonic acid in methylene chloride containing molecular sieves which remove any water present.
  • the reaction mixture is cooled to -50"C and then compound
  • compound 32a or 112 can be used in place of compound 32.
  • Compound 112 is analagous to compound 32a but has a 4,6-p-methoxy- benzylidine protecting group on the galactose [prepared from compound 28 using (CH 3 0) 2 CH-C 6 H 4 -p-OCH 3 ] instead of the benzylidine protecting group of compound 32a.
  • the benzoyl groups on disaccharide 102 are removed under Zemplen conditions (NaOMe/MeOH) to provide for disaccharide 103 having free hydroxyl groups at the 2,3 positions of the galactose.
  • Disaccharide 103 is sulfated in DMF at about -30° to -50°C using about 1.1 to 1.5 equivalents of sulfur trioxide/pyridine complex to provide for the 3-sulfated disaccharide 104 as the pyridinium salt.
  • This product contains some 2-sulfated and some 2,3-disulfated material which can be separated by chromatography. The selectivity of the sulfating step is improved by the use of colder temperatures.
  • compound 103 can be chloroacetylated under typical conditions to provide for a mixture of the 2- and 3-chloroacetyl protecting groups.
  • This mixture can be separated by chromatography and the resulting purified components can be used to prepare 2- or 3-sulfated products selectively, or for that matter, the 2-phosphorylated and 2-OCHR 18 COOH substituents (discussed below) , both of which find utility in this invention as immunomodulating compounds.
  • a differentially protected Lewis°-OR structure can be prepared by combining compound 5 with compound 31 under suitable conditions to provide for a fully blocked Lewis°-OR structure having a 3- chloroacetyl blocking group and a 2-benzoyl blocking group on the galactose unit.
  • This compound can then be selectively deblocked at the 3-position of the galactose and then sulfated in the manner described above to provide for the 3-sulfated blocked structure.
  • deprotection of this product may lead to the generation of by products (e.g. , the 2-NH 2 of the GlcNAc) . If necessary, these by products can be separated by chromatography.
  • Figure 3B illustrates one method for preparing the 6-sulfate Lewis°-OR compound.
  • compound 107 is prepared from compound 28 by treating compound 28 with p-anizaldehyde dimethylacetal under conditions otherwise identical to the preparation of compound 29. The resulting product is benzoylated under conditions similar to those employed to prepare compound 32. This compound is then ring opened using sodium cyanoborohydride in acidic medium in tetrahydrofuran which yields the free 4-OH derivative which, in turn, is acetylated under typical conditions to provide for compound 107. Compound 107 is then converted to the 1-cc-bromo derivative using Norberg 26 conditions (bromine, tetraethylammonium bromide at 0"C in methylene chloride) to provide for compound 108.
  • Norberg 26 conditions bromine, tetraethylammonium bromide at 0"C in methylene chloride
  • Compound 108 is then combined with compound 101 in the presence of methylene chloride, silver carbonate (about 3 equivalents) , silver triflate (about 0.1 equivalents) and molecular sieves.
  • the reaction is conducted at about 0°C to room temperature for about 5 to about 16 hours to provide for the fully protected Lewis°-0R derivative, compound 109.
  • the p-methoxybenzyl blocking group at the 6-position of the galactose unit is selectively removed by contacting compound 109 with dichlorodicyanoquinone (DDQ) in methylene chloride and a trace of water to provide for compound 110.
  • DDQ dichlorodicyanoquinone
  • Compound 110 is then sulfated at the 6-position of the galactose in DMF at about -50" to 0°C using about 1.1 to 1.5 equivalents of sulfur trioxide/pyridine complex to provide for the 6-sulfated blocked Lewis°-OR as the pyridinium salt.
  • Figure 4 illustrates one method for the preparation of intermediates useful in the preparation of 3-sulfated LacNAc-OR compounds. Specifically, in Figure 4, compound 7 and approximately 1.6-1.7 equivalents of compound 32a are dissolved in dichloromethane containing molecular sieves to which is adde ⁇ about 1 equivalent (based on compound 7) of 2,6- di-t-butyl-4-methylpyridine. The reaction is stirred for 30 minutes at room temperature and then cooled to -50°C.
  • anhydrous toluene solution containing approximately a slight excess (e.g., about 1.2 equivalents) of silver trifluoromethanesulfonate is then added to the solution and the reaction was allowed to warm to -15°C over 2 hours and maintained at that temperature for an additional 5 hours.
  • reaction system was worked up to provide a crude product of compound 42. This is then purified by conventional techniques such as column chromatography using silica gel and toluene-ethyl acetate (1:1) as the eluant.
  • the benzoyl groups on the protected LacNAc-OR 42 are removed under Zemplen conditions to provide for the LacNAc-OR derivative having free hydroxyl groups at the 2,3 positions of the galactose.
  • This disaccharide is sulfated in DMF at about -30° to -50 ⁇ C using about 1.1 to 1.5 equivalents of sulfur trioxide/pyridine complex in the manner described above to provide for the blocked 2-hydroxy-3-sulfated LacNAc-OR as the pyridinium salt.
  • This product contains some 2-sulfated and some 2,3-disulfated material which can be separated by chromatography. The selectivity of the sulfating step is improved by the use of colder temperatures.
  • the 2,3-dihydroxy disaccharide can be chloroacetylated under typical conditions to provide for a mixture of the 2- and 3-chloroacetyl protecting groups.
  • This mixture can be separated by chromatography and the resulting purified components can be used to prepare 2- or 3-sulfated products selectively.
  • Figure 4 also illustrates a differentially blocked LacNAc-OR structure which can be used to prepare 3-sulfated structures.
  • compound 7 and compound 33 are combined to form compound 37. This is accomplished by dissolving compound 7 and approximately 1.5 equivalents of compound 33 in dichloromethane containing molecular sieves to which is added about 1 equivalent (based on compound 7) of 2,6-di-t-butyl-4-methylpyridine. The reaction is stirred for 30 minutes at room temperature and then cooled to -50"C.
  • anhydrous toluene solution containing approximately a slight excess (e.g., about 1.2 equivalents) of silver trifluoromethane sulfonate is then added to the solution and the reaction is allowed to warm to -15°C over 2 hours and maintained at that temperature for an additional 5 hours.
  • the molecular sieves are removed by filtration by passing through celite and the recove d solution is quenched by addition to a saturated sodium bicarbonate solution.
  • the organic extract is then washed with water, with aqueous 0.5N HCl, and then with water.
  • the organic solution is then dried and concentrated in vacuo to provide a crude product, compound 37. This is then purified by conventional techniques such as column chromatography using silica gel and hexane-ethyl acetate (1:1) as the eluant.
  • Compound 37 is then selectively deblocked at the 3-position of the galactose and then sulfated in the manner described above to provide for the 3- sulfated blocked structure.
  • deprotection of this product may lead to the generation of by products (e.g. , the 2-NH 2 of the GlcNAc) . If necessary, these by products can be separated by chromatography.
  • the 6-sulfate derivative of LacNAc-OR is prepared in the manner described above for the 6-sulfate of Lewi ⁇ °-OR with the exception that compounds 7 or 9 are used in place of compound 101 in this reaction.
  • Blocked Lewis°-OR and blocked LacNAc-OR compounds having a 2, 3- or 6-hydroxyl group (e.g., compound 103) on the galactose unit can be can be converted to the 2-, 3-, 6-phosphate group on the galactose by reaction with diphenylphosphorochloridate and 4-dimethylaminopyridine (1:1) in pyridine at 0"C 16 .
  • the solution is allowed to warm to room temperature over 0.5 hours and stirred for 15 hours.
  • the resulting compound is then hydrogenated under conventional conditions (first with H 2 in EtOH with Pd on carbon for 15 hours and then with H 2 in EtOH with Pt0 2 for 3 hours) to provide for the phosphate derivative at the 2-, 3-or 6-position of galactose.
  • blocked Lewis c -OR and blocked LacNAc-OR compounds having a 2-, 3- or 6- hydroxyl group (e.g., compound 103) on the galactose unit can then be alkylated by first adding an appropriated base (e.g., silver oxide, barium hydroxide, sodium hydride) and then adding benzyl bromoacetate (BrCH 2 COOBn) or other similar acetates (e.g., BrCHR 18 ,COOBn — where R 18 , is alkyl of from 1 to 7 carbon atoms or -COOBn) to the reaction medium in an appropriate solvent such as DMF.
  • an appropriated base e.g., silver oxide, barium hydroxide, sodium hydride
  • BrCH 2 COOBn benzyl bromoacetate
  • R 18 is alkyl of from 1 to 7 carbon atoms or -COOBn
  • the benzyl ester(s) is (are) readily removed by conventional hydrogenation techniques which additionally removes the other benzyl protecting groups and the benzylidine protecting group.
  • Treatment with sodium methoxide/methanol provides for a -OCH 2 COOH (or -OCHR 18 COOH where R 18 is alkyl of from 1 to 7 carbon atoms or -COOH) substituted to the 2-, 3-, 6-position of galactose.
  • the 2,6 positions of the GlcNAc unit can be modified prior to coupling so as to provide for Lewis c -OR and LacNAc-OR structures modified at these positions which are then further modified in the manner described above to prepare the sulfated, phosphorylated or -CHR 18 COOH derivatives.
  • function- alization of the GlcNAc unit at the 2,6 position is generally at a point in the synthesis where the to-be formed functional group does not interfere with any of the further intended reactions. For example, if an R.
  • the functional group in GlcNAc-OR would interfere with the coupling reaction with the blocked galactose, then the functional group can either be introduced at the disaccharide level or blocked at the monosaccharide level (e.g., 2-amino groups are conventionally blocked with as the N-trifluoroacetamido or the N-phthalimido group) and later deblocked.
  • Modification at the 2-position of GlcNAc can be accomplished by a variety of ways.
  • the known 8,9 2-azido-2-deoxy-glucose-OR compound prepared, for example, by azidonitration of 4,5,6- triacetylglucal
  • a removable protecting group i.e., Si(C 6 H 5 ) 2 tBu
  • an appropriate blocked galactose compound in the manner described above to provide for the blocked 3Gal(l ⁇ 3)GlcN 3 -OR and 0Gal(l ⁇ 4)GlcN 3 -OR derivatives which are readily separated by conventional techniques.
  • the azido group is reduced to an amino group which can be protected, for example, as N-trifluoroacetamido.
  • the trifluoroacetamido group is removed at the appropriate point in the synthe is thereby unmasking the amino group.
  • the -NH 2 group can be reacted, using conventional techniques: with a carboxylic acid, anhydride or chloride to provide for amides.
  • the desired acid can be activated, as reported by Inazu et al 37 and then reacted with the amino group.
  • This compound is then converted to the 3,4,6-tribenzyl 1,2-ortho ester of glucose using conventional techniques.
  • the 1,2-ortho ester of the resulting compound is then opened by conventional techniques to provide a protected glycosyl donor such as the l- ⁇ -bromo-2-acetyl-3,4,6-tribenzyl derivative of glucose.
  • This 1- ⁇ -bromo derivative is then converted to the glycoside (-OR) by conventional techniques and the 2-acetyl group is then removed.
  • the 2-position is now ready for formation of the 2-deoxy by conventional methods such as first treating with carbon disulfide and methyl iodide in the presence of one equivalent of a base to form the -C(S)SCH 3 derivative, followed by reaction with tributyltin hydride) or for the preparation of the 2-alkoxy.
  • the remaining protecting groups are removed so as to provide for 2-deoxyglucose glycoside or a 2-alkoxyglucose glycoside which can then be derivatized in the manner described above and illustrated in Figure 1 without the need to form the aglycon.
  • the 6-azido derivatives of GlcNAc-OR can be prepared in the manner described in Figure 5. Specifically, GlcNAc-OR, compound 87, is converted to the p-methoxybenzylidine blocked compound 88 by reaction with (CH 3 0) 2 CH-C 6 H 4 -p-OCH 3 . This compound is then protected at the 3-hydroxyl position by reaction with 4-CH 3 0-C 6 H 4 -CH 2 Br to provide for compound 89 where X is 4-CH 3 0-C 6 H 4 -CH 2 -. Compound 89 is partially deprotected at the 4 and 6 positions by reaction with acetic acid (AcOH) in water at about 45°C to provide for compound 90.
  • AcOH acetic acid
  • the 6-mesylate, compound 91 is prepared by reacting compound 90 with mesyl chloride in pyridine (MsCl/py) .
  • the 6-azido derivative, compound 92 is then formed by reaction with sodium azide in dimethylformamide (DMF) and removal of the 3-blocking group with dichlorodicyanoquinone (DDQ) yields compound 93.
  • DMF dimethylformamide
  • DDQ dichlorodicyanoquinone
  • the 6-mesyl compound 91 can also be derivatized to any of a number of 6-substituents including alkoxy substituents, and the like by well known chemistry.
  • the 6-azido compound 92 can be derivatized to the 6-amino at an appropriate point in the synthesi ⁇ of the Lewis c -OR or LacNAc-OR analogues in the manner described above.
  • the -NH 2 group can be reacted, using conventional techniques, with: a carboxylic acid, anhydride or chloride to provide for amides.
  • the desired acid can be activated, as reported by Inazu et al 37 and then reacted with the amino group.
  • a cyclic carbonate such as ethylene carbonate or propylene carbonate which ring opens upon reaction with the amine to form a carbamate group having an HO-alkylene-OC(0)NH- substituent where alkylene is from 2 to 4 carbon atoms as reported by Wollenberg et al. 39 , U.S. Patent No. 4,612,132, with a chloroformate [i.e., C1C(0)0R 7 ] in the manner disclosed by Greig et al. 40 .
  • the 6-alkoxy derivatives of GlcNAc can be prepared in the manner described in Figure 6. Specifically, GlcNAc-OR, compound 87, is reacted with C 6 H 5 CH(OCH 3 ) 2 in an acidic medium in acetonitrile to provide for the 4,6-diprotected benzylidine compound 94. In turn, compound 94 can be reacted with benzyl (Bn) bromide and sodium hydride in the presence of dimethylformamide at around 0°C to provide for a benzyl protecting group at the 3-position, i.e., compound 95. Deprotection at the 4,6 positions by contacting compound 95 with acetic acid and water at about 80°-90 ⁇ C provides for compound 96.
  • compound 94 can be reacted with [C 6 H 5 C(0)] 2 0 in pyridine to provide for a benzoyl protecting group (Bz) at the 3-position, i.e., compound 99.
  • Bz benzoyl protecting group
  • Reaction of compound 99 with N-bromo- succinimide in carbon tetrachloride yields the 6-bromo compound 100.
  • Compound 100 can be reacted with tributyltin hydride [(Bu) 3 SnH] in toluene to provide for the 6-deoxy compound 100b which after conventional deprotection of the benzoyl groups with ⁇ odium methoxide in methanol gives the 6-deoxy compound 100c.
  • the 6-SR 6 compounds are prepared from the 6- mesyl derivative, compound 91, by reaction with potassium thioacetate, CH 3 C(0)S " K + , to give the thioacetate derivative at the 6-position. This derivative is then treated with mild base to produce the 6-SH derivative.
  • the 6-SH can be reacted with an alkyl halide (e.g., CH 3 Br) to provide the -SR 6 derivatives which, in turn, can be partially or fully oxidized to the 6-sulfone or the 6-sulfoxide derivatives, -S(0)R 6 and -S(0) 2 R 6 where R 6 is alkyl of from 1 to 4 carbon atoms.
  • an alkyl halide e.g., CH 3 Br
  • Figure 7 illustrates the preparation of 3- hydroxy or 4-hydroxy blocked GlcNH 2 -0R where the amino group is protected as an N-phthalimido group.
  • compound 13 is prepared by the methods described above. This compound is then deacetylated by conventional techniques (sodium methoxide/methanol) to provide for compound 14 which is then benzylidenated under conventional techniques to provide compound 66. Compound 66 is then treated with benzyl chloride and sodium hydride in dimethylformamide at about -20°C to 20 ⁇ C to provide for compound 67. The benzylidine group of compound 67 is then removed with 80% aqueous acetic acid at about 80 ⁇ C for about 1-4 hours to provide for compound 68. This compound is then selectively acetylated at the 6-po ⁇ ition by use of approximately equimolar amounts of acetyl chloride/pyridine in dichloromethane at about -10°C to provide for compound 69.
  • compound 69 is useful in preparing LacNH 2 derivatives whereas compound 66 is useful in preparing Lewis-OR derivatives.
  • the 2- or 3-sulfate, phosphate or -OCHR 18 C00H substituted LacNAc structures can be converted to the 6-sialyl derivatives by use of the known /3Gal(1 ⁇ 4)jSGlcNAc ⁇ (2 ⁇ 6)sialyltransferase in a manner similar to that described in the example ⁇ herein below.
  • the 6-sialyl derivatives of Lewis c -OR can be prepared in a manner similar to that of Ratcliffe et al. 12,23 making the appropriate modifications to the blocking groups to allow for subsequent substitution with a sulfate, phosphate or -CHR 18 COOH group at the 2- or 3- positions of galactose.
  • the modified Lewis c -YR and LacNAc-YR compounds disclosed herein affect the cell mediated immune respon ⁇ e in a number of way ⁇ . Specifically, these compounds can inhibit the ability of the immune response to become educated about a specific antigen when the compound is administered simultaneously with the first exposure of the immune system to the antigen.
  • the modified Lewis c -YR and LacNAc-YR compounds disclosed herein can inhibit the secondary immune response to an antigen in a sen ⁇ itized mammal when administered after second or later exposures of the immune system to the same antigen. Additionally, the modified Lewis 0 -YR and LacNAc-YR compounds disclosed herein can induce tolerance to antigens when administered at the time of second or later expo ⁇ ures of the immune system to the antigen.
  • Tl suppression of the inflammatory component of the secon ⁇ -_ry immune response by the modified Lewis°-YR and LacNAc-YR compounds disclosed herein requires administering such compounds after initiation of the mammal's secondary immune response but at or prior to one-half the period required for maximal antigen induced inflammation. This criticality is disclosed in U.S. Patent Application No. 07/988,518 filed concurrently herewith as Attorney Docket No.
  • the modified Lewis°-OR and LacNAc-OR compounds are preferably administered to the patient at least about 0.5 hours after antigen exposure, more preferably, at least about 1 to 10 hour after antigen exposure, and still more preferably, from about at least about 1 to 5 hour ⁇ after antigen exposure.
  • the modified Lewis c -YR and LacNAc-OR glycosides disclosed herein are effective in suppressing cell-mediated immune respon ⁇ e ⁇ including cell-mediated immune response to an antigen (eg. the inflammatory component of a DTH response) as well as in suppressing cell-mediated inflammatory respon ⁇ e ⁇ to injury (e.g., lung injury) when admini ⁇ tered at a dosage range of from about 0.5 mg to about 50 mg/kg of body weight, and preferably from about 0.5 to about 5 mg/kg of body weight.
  • the specific dose employed is regulated by the particular cell-mediated immune response being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the adverse immune response, the age and general condition of the patient, and the like.
  • the modified Lewis c -YR or LacNAc-YR analogues are generally administered parenterally, such as intranasally, intrapulmonarily, transdermally and intravenou ⁇ ly, although other form ⁇ of administration are contemplated.
  • administering In addition to providing suppression of a mammal's secondary immune response to an antigen, administration of the modified Lewis c -YR and LacNAc-YR compounds disclosed herein also imparts a tolerance to additional challenges from the same antigen provided that the compound is administered during the critical period discussed above. In this regard, re-challenge by the same antigen weeks after administration of the modified Lewis c -YR or LacNAc-YR compound ⁇ re ⁇ ults in a significantly reduced immune respon ⁇ e.
  • the modified Lewis c -YR and LacNAc-YR compounds disclosed herein simultaneou ⁇ ly with first exposure to an antigen (i.e., a non-sensitized mammal) imparts suppression of a cell- mediated immune response to the antigen and tolerance to future challenges with that antigen.
  • an antigen i.e., a non-sensitized mammal
  • the term "reducing sensitization” means that the modified Lewis c -YR or LacNAc-YR compound, when administered to a mammal in an effective amount along with a sufficient amount of antigen to induce an immune response, reduces the ability of the immune system of the mammal to become educated and thus ⁇ ensitized to the antigen administered at the same time as the modified Lewis c -YR or LacNAc-YR compound.
  • an "effective amount" of this compound is that amount which will reduce sensitization (immunological education) of a mammal, including humans, to an antigen administered simultaneously as determined by a reduction in a cell-mediated response to the antigen such as DTH responses as te ⁇ ted by the footpad challenge test.
  • the reduction in sensitization will be at least about 20% and more preferably at least about 30% or more.
  • modified Lewis c -YR and LacNAc-YR compounds disclosed herein are effective in reducing sensitization when administered at a dosage range of from about 0.5 mg to about 50 mg/kg of body weight, and preferably from about 0.5 mg to about 5 mg/kg of body weight.
  • sensitization is regulated by the sensitization being treated as well a ⁇ the judgement of the attending clinician depending upon the age and general condition of the patient and the like.
  • "Simultaneou ⁇ " admini ⁇ tration of the compound with the antigen with regard to inhibiting sensitization means that the compound is administered once or continuously throughout a period of time within 3 hours of the administration of an antigen, more preferably the compound is administered within 1 hour of the antigen.
  • compositions suitable for use in the parenteral administration of an effective amount of a Lewis c -YR or a LacNAc-YR compound comprise a pharmaceutically inert carrier ⁇ uch as water, buffered saline, etc and an effective amount of a modified Lewi ⁇ c -YR or LacNAc-YR compound ⁇ o as to provide the above-noted dosage of these compounds when administered to a patient.
  • suitable pharmaceutical composition ⁇ can additionally contain optional components such as a preservative, etc
  • compositions can include oral compositions, transdermal compositions or bandages etc. , which are well known in the art.
  • IR-120 resin amberlite resin available from Rohm & Haas, Philadelphia, PA
  • IR-C50 resin (H * form) ion exchange resin IR-C50 (H + form) available from Rohm & Haas, Philadelphia, PA
  • the following example ⁇ are divided into two parts.
  • the first part (part I) relates to the synthetic procedures to make the recited compounds whereas the second part (part II) relates to the biological results.
  • the reaction mixture is drained into a clean 20 L polyethylene pail.
  • the 50 L reactor is charged with 15 L of saturated ⁇ odiu carbonate solution.
  • the 20 L polyethylene pail is slowly transferred into the slowly stirring carbonate solution at such a rate that the gas evolution is not overly vigorous. Stir the solution for 20 minutes then increase the rate of stirring. When gas evolution cease ⁇ bubble air through the entire ⁇ olution for 24-36 hours. Drain the organic layer into a clean 20 L polyethylene pail and store in a hood. Extract the sodium carbonate solution with 3-5 L of dichloromethane and drain thi ⁇ solution into the same 20 L polyethylene pail.
  • the organic ⁇ olution can be filtered using a 4 L vacuum filtration set and the filtrate evaporated under reduced pre ⁇ sure on the 20 L rotovap.
  • 7 L of methanol is introduced into the rotavap flask and the residue heated with the rotavap bath till the residue dis ⁇ olves in the warm methanol.
  • the flask is rotated and allowed to cool. Cool ice water is added to the rotavap bath and the flask slowly rotated for several hours.
  • the flask is removed from the rotovap and the white crystalline product filtered u ⁇ ing a 4 L vacuum filtration set.
  • the benzyl 2,3,4,6-tetra-O-acetyl- ⁇ -D- thiogalactopyranoside (-1.3 kg) is charged into a clean dry 20 L reactor with stirring motor and 7 L of dry methanol is added to dis ⁇ olve the material.
  • the ⁇ olution i ⁇ treated with 3 g of freshly surfaced sodium and stirred for two hours.
  • the reaction is checked by t.l.c on silica gel using a retained sample of the benzyl 2,3,4,6-tetra-O-acetyl- ⁇ -D-thiogalactopyranoside with 80:20 ethyl acetate: methanol (v/v) the eluant.
  • Benzyl-4,6-0-benzylidene-3-D-thiogalacto- pyranoside (10 g) was dissolved in 100 mL dichloromethane and 6.35 g of pyridine was added. To the solution was added 9 g of benzoyl chloride in dropwise fashion and after 1 hour, 50 mg of dimethylaminopyridine was added to the solution and the mixture wa ⁇ stirred for. an addition 2 to 4 hour ⁇ . The progre ⁇ of the reaction wa ⁇ checked by t.l.c on silica gel.
  • Benzyl-4,6-0-benzylidene-2,3-di-0-benzoyl- 0-D-thiogalactopyranoside (compound 32) was i ⁇ olated by quenching the reaction mixture into ⁇ aturated sodium bicarbonate solution and washing the organic extract with water, 5% copper sulfate solution, water, drying and evaporating the solvent. The re ⁇ idue was crystallized from isopropanol to give 10.7 g of compound 32.
  • Example 3 Synthesis of 8-methoxycarbonyloctyl-2- acetamido-4,6-di-0-benzylidene-2-deoxy- ⁇ -D-glucopyrano ⁇ ide (Compound 5)
  • a 20L glass reactor wa ⁇ charged with 8 L of dichloroethane, 1 L of acetic anhydride and 1 kg of anhydrou ⁇ ⁇ odium acetate.
  • To the ⁇ tirring mixture was added 1 kg of glucosamine hydrochloride and the mixture was brought to reflux.
  • a further 3.5 L of acetic anhydride was added dropwise to the refluxing ⁇ olution over 3-4 hour ⁇ and the ⁇ olution maintained at reflux for 36 hours.
  • reaction mixture was filtered through a buchner funnel of silica and the organic layer washed twice with water, twice with a 5% solution of potas ⁇ ium iodide and twice with a saturated solution of sodium bicarbonate. The solution was dried over sodium sulphate and evaporated to dryness. The residue was taken up in anhydrou ⁇ methanol and treated with 1 g of freshly cut sodium then stirred at room temperature overnight.
  • the reaction appears complete the mixture was neutralized with triethylamine and quenched into several volumes of ice water, extracted into dichloromethane and backwashed several times with water.
  • the organic layer was dried over sodium sulphate, evaporated to dryness and taken up in hot isopropanol. After cooling 8- methoxycarbonyloctyl-2-acetamido-4,6-0-benzylidene-2- deoxy- ⁇ -D-glucopyrano ⁇ ide precipitates. It is filtered and dried to yield 106 g of product. 1 H-n.m.r.
  • Example 8 Synthe ⁇ i ⁇ of 8-methoxycarbonyloctyl 2- acetamido-4-O-(2*-O-benzoyl-4• ,6'-O- benzylidene-3 ' -O-chloroacetyl- ⁇ -D- galactopyranosyl)-6-0-benzyl-2-deoxy-3- O-p-methoxybenzyl- ⁇ -D-gluco-pyranoside
  • Ethyl 2-deoxy-2-phthalamido-3,4,6-tri-0-acetyl-/3- D-glucopyrano ⁇ yide (compound 13) from example 17 wa ⁇ taken up in 100 mL of dry methanol and treated with 100 mg of ⁇ odium metal. The ⁇ olution wa ⁇ ⁇ tirred at room temperature for 24 hour ⁇ and then neutralized with Ambe lite [R-120(H+) ] re ⁇ in, filtered, and evaporated to dryne ⁇ in vacuo. This compound was u ⁇ ed in the preparation of compound 15 and compound 66.
  • Example 13 Synthesis of Ethyl 2-deoxy-2- phthalimido-6-O-benzyl- ⁇ -D- glucopyrano ⁇ ide (Compound 15)
  • Compound 14 (2.1 g, 6.23 mmol) was taken up in 100 mL of toluene. To it was added bis(tributyl tin) oxide (2.22 mL, 4.35 mmol) and tetrabutylammonium bromide (0.983 g, 3.05 mmol). The mixture was heated at 150°C for 4 hours and then toluene (50 mL) was distilled off from the mixture. The reaction mixture was cooled to room temperature and benzyl bromide (2.17 -mL, 18.27 mmol) wa ⁇ added and the reaction heated to 110°C for 36 hour ⁇ .
  • Example 15 Synthesis of Ethyl 6-0-acetyl-3-0- benzyl-2-deoxy-2-phthalimido- ⁇ -D- glucopyrano ⁇ ide (compound 69)
  • a ⁇ olution of ethyl 2-deoxy-2-phthalimido-j8-D- glucopyranoside (compound 14) from Example 12 was taken up in dry aoetonitrile (100 mL) and treated with benzaldehyde dimethylacetal (9.6 g) and a catalytic amount of p-toluenesulphonic acid (100 mg) .
  • the mixture was stirred for 17 hours at room temperature and then neutralized to pH 7 with triethylamine.
  • the title compound was prepared by first generating 8-methoxycarbonyloctyl 3-0-(2,3-di-0- benzoyl-4,6-0-benzylidene- / S-D-galactopyrnosyl)-2- acetamido-4,6-O-benzylidene-2-deoxy-0-D-glucopyranoside which, in turn, was generated by coupling the 8- methoxycarbonyloctyl 2-acetamido-4,6-0-benzylidene-2- deoxy-3-D-glucopyrano ⁇ ide and compound 32, the ⁇ ynthe ⁇ i ⁇ of which is exemplified in example 2 above.
  • the 8-methoxycarbonyloctyl 2-acetamido-4,6-0- benzylidene-2-deoxy-)3-D-glucopyranoside can be prepared by reacting N-acetylglucosamine-OR (e.g., compound 4 in Figure 1) with about 1.5 equivalent ⁇ of C 6 H 5 CH(OCH 3 ) 2 in an acidic (p-toluene ⁇ ulfonic acid) aoetonitrile or dimethylformamide (DMF) medium at from about 0° to about 50°C over 6-48 hours to provide for the 4,6-0- protected benzylidine compound.
  • N-acetylglucosamine-OR e.g., compound 4 in Figure 1
  • an acidic (p-toluene ⁇ ulfonic acid) aoetonitrile or dimethylformamide (DMF) medium at from about 0° to about 50°C over 6-48 hours to provide for the 4,6-0- protected benzylidine compound.
  • Coupling of compound 32 with the 8-methoxycarbonyloctyl 2-acetamido-4,6-0-benzylidine-2- deoxy-3-D-glucopyrano ⁇ ide can be achieved by first combining about 1 equivalent of N- iodosuccinimide with about 1 equivalent of trifluoromethanesulfonic acid in methylene chloride containing molecular sieves which remove any water present. The reaction mixture is cooled to -50"C and then compound A is added followed by the addition of approximately, 1 to 1.1 equivalents of compound 32 (based on compound A) . When large amounts of trifluoromethanesulfonic acid are employed, the reaction is preferably cooled to -50°C prior to the addition of the trifluoromethanesulfonic acid.
  • reaction is allowed to equilibrate to about -20° to 0°C over about 1-3 hour ⁇ .
  • the reaction solution is then quenched by cooling to -50° followed by the addition of triethylamine until neutral pH is reached.
  • the solution i ⁇ filtered through Celite and then washed with a saturated sodium bicarbonate solution and water.
  • the organic layer was dried and ⁇ tripped in vacuo to provide for 8-methoxycarbonyloctyl 3-0-(2,3-di-0-benzoyl-4,6-0-benzylidene-3-D- galactopyrano ⁇ yl)-2-acetamido-4,6-0-benzylidine-2- deoxy-/3-D-glucopyrano ⁇ ide ("compound B”) .
  • the benzoyl group ⁇ on compound B can be removed under Zemplen conditions (NaOMe/MeOH) to provide for 8-methoxycarbonyloctyl 3-0-(4,6-0- benzyl idene-3-D-galactopyrano ⁇ yl) -2-acetamido-4 , 6-0- benzylidene-2-deoxy-3-D-glucopyranoside ("compound C”) .
  • Example 17 The product of Example 17 (800 mg, 0.96 mmol) was dis ⁇ olved in methanol (10 mL) containing 5% palladium on carbon (800 mg) and was stirred under hydrogen (1 atmosphere) for 5 hours at room temperature. Catalyst was removed by filtration, washed with methanol (500 mL) and the solvent was evaporated to dryness. The residue was then purified by chromatography on silica gel using dichloromethane- methanol-water-pyridine (80:20:2:0.2) as eluant. The title compound (488 mg, 77.5%) wa ⁇ obtained a ⁇ a white ⁇ olid after BioGel P-2 (200-400 me ⁇ h) filtration and conver ⁇ ion into its sodium salt.
  • Example 18 wa ⁇ prepared in a ⁇ imilar manner except that ⁇ ulfation wa ⁇ conducted with 6 equivalent ⁇ of ⁇ ulfur trioxide/pyridine complex and the reaction wa ⁇ conducted at room temperature for 24 hour ⁇ .
  • the resulting product was a mixture of 2-, 3-sulfate and predominantly 2,3-disulfate.
  • the mixture wa ⁇ purified by chromatography in the manner described and ion exchange of the resulting product provided for the 2,3-disulfate of Lewis c -0R a ⁇ the disodium salt.
  • the 2-sulfate of Lewis c -OR wa ⁇ prepared from compound C by benzoylation under conditions described above.
  • the resulting product contained predominantly the 3-benzoyl group on the galactose unit and a small amount of the 2-benzoyl and 2,3-dibenzoyl derivatives.
  • the products were separated by chromatography on silica gel to provide for both the 2-benzoyl and the 3-benzoyl derivatives as pure products.
  • the 3-benzoyl derivative was sulfated in the manner described above and then deprotected to provide for the 2'-sulfo-Lewis c -OR which upon ion exchange as de ⁇ cribed above provided for the sodium salt of this product.
  • the 3'-sulfo-LacNAc derivative was prepared from compound 42 (prepared in Example 6) wherein the dibenzoyl groups are removed via Zemplen conditions (sodium methoxide/methanol) and then sulfated under the conditions described above to provide for 3'-sulfo-Lewis c -OR.
  • DTH inflammatory respon ⁇ es were measured using the mouse footpad swelling as ⁇ ay as described by Smith and Ziola 14 . Briefly, groups of Balb/c mice were immunized with 100 ⁇ g of the OVA antigen [Albumin, chicken egg, Sigma, St. Louis, Mis ⁇ ouri] containing 20 ⁇ g of the DDA adjuvant (dimethyldioctadecylammonium bromide) which ha ⁇ been ⁇ hown to induce a strong inflammatory DTH response. Seven days later, each group of mice was footpad-challenged with 20 ⁇ g of OVA antigen. The re ⁇ ulting inflammatory footpad ⁇ welling was measured with a Mitutoyo Engineering micrometer 24 hours after challenge.
  • OVA antigen Albumin, chicken egg, Sigma, St. Louis, Mis ⁇ ouri
  • mice received 100 ⁇ g of the following oligosaccharide glycosides 5 hours after foot-pad challenge with the OVA antigen.
  • Compound D Sialyl Lewis*-OR ( ⁇ Neu5Ac(2-*3)/3Gal(1-3) ⁇ [ ⁇ -L-Fuc(1-4) ]-/3GlcNAc-OR]
  • mice are those mice which receive one of the above compounds in addition to the antigen.
  • Untreated Mice are those mice which do not receive any of the above compounds.
  • Background (bkg) swelling is that level of swelling observed in mice immunized with PBS alone without antigen or compound and challenged with antigen.
  • LPS lipopolysaccharide caused lung injury is measured by weighing the lungs of sacrificed mice 24 hours after mice are given LPS intranasally. Briefly, groups of 8-10 week old Balb/c mice were sensitized with 5 ⁇ g /mouse of LPS in 50 ⁇ l of PBS intranasally under light anesthe ⁇ ia.
  • mice are anethe ⁇ itized with Metofane (Pitman-Moore Ltd. , Mi ⁇ i ⁇ sauga, Ontario, Canada) and a 50 ⁇ l drop of compound is placed on the nares of the mou ⁇ e and is inhaled.
  • Metofane Pane-Moore Ltd. , Mi ⁇ i ⁇ sauga, Ontario, Canada
  • Lewi ⁇ c -OR in 200 ⁇ l of PBS is given to the mouse intravenously. After 24 hours, 48 hours or 72 hours, different groups of mice are ⁇ acrificed and the lungs removed and weighed. The weight of the lungs of mice treated with 3-sulfo-Lewi ⁇ c -0R are compared against control (i.e., mice treated with LPS but to which 3- sulfo-Lewis c -OR has not been administered) .
  • the percent reduction is measured by subtracting from 100 the following: The fraction derived by a numerator whose value is the weight of the treated lungs subtracted from the weight of normal lungs (lungs from mice not exposed to LPS, 3-sulfo-Lewi ⁇ c -OR) , and whose denominator whose value is the weight of the control lungs (mice that received only LPS) subtracted from the weight of normal lungs and multiplying the resulting fraction by 100.

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Abstract

L'invention décrit de nouveaux analogues de LewisC et de LacNAc, des compositions pharmaceutiques contenant ces analogues, des procédés pour leur préparation ainsi que des procédés pour leur utilisation.
PCT/US1993/004995 1992-05-26 1993-05-26 COMPOSES LEWISC ET LacNAc MODIFIES A EFFET IMMUNOSUPPRESSEUR ET INDUCTEUR DE TOLERANCE WO1993024506A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0714903A1 (fr) * 1994-10-10 1996-06-05 Hoechst Aktiengesellschaft Nouveaux carbohydrat conjugués utilisés comme inhibiteurs d'adhésion cellulaire
WO1997018222A2 (fr) * 1995-11-13 1997-05-22 Glycomed, Inc. Nouveaux glycosides oligosaccharidiques ayant des proprietes immunosuppressives et tolerogenes chez les mammiferes
EP0795560A1 (fr) * 1994-12-01 1997-09-17 Seikagaku Corporation Fraction d'oligosaccharide de sulfate de keratane et medicament la contenant
WO1998004270A1 (fr) * 1996-07-31 1998-02-05 Roc Utilisation d'un oligosaccharide en tant qu'immunomodulateur dans une composition dermato-cosmetique
US6436911B1 (en) 1999-07-21 2002-08-20 Seikagaku Corporation IL-12 production inhibitor
US6562954B1 (en) 1999-01-07 2003-05-13 Seikagaku Corporation Method for producing oligosaccharide, and novel oligosaccharide and pharmaceutical composition containing the same
WO2005061523A1 (fr) * 2003-12-23 2005-07-07 Progen Industries Limited Composes mimetiques du glycosaminoglycane (gag)
US7332480B2 (en) * 1992-05-01 2008-02-19 Yeda Research And Development Co. Ltd. Compositions for the regulation of cytokine activity

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5079235A (en) * 1986-12-15 1992-01-07 Burroughs Wellcome Co. Antiviral compounds
US5079353A (en) * 1987-12-02 1992-01-07 Chembiomed, Ltd. Sialic acid glycosides, antigens, immunoadsorbents, and methods for their preparation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5079235A (en) * 1986-12-15 1992-01-07 Burroughs Wellcome Co. Antiviral compounds
US5079353A (en) * 1987-12-02 1992-01-07 Chembiomed, Ltd. Sialic acid glycosides, antigens, immunoadsorbents, and methods for their preparation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0643718A4 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7332480B2 (en) * 1992-05-01 2008-02-19 Yeda Research And Development Co. Ltd. Compositions for the regulation of cytokine activity
EP0714903A1 (fr) * 1994-10-10 1996-06-05 Hoechst Aktiengesellschaft Nouveaux carbohydrat conjugués utilisés comme inhibiteurs d'adhésion cellulaire
US5858994A (en) * 1994-10-10 1999-01-12 Hoechst Aktiengesellschaft Carbohydrate conjugates as inhibitors of cell adhesion
EP0795560A1 (fr) * 1994-12-01 1997-09-17 Seikagaku Corporation Fraction d'oligosaccharide de sulfate de keratane et medicament la contenant
JP2010046070A (ja) * 1994-12-01 2010-03-04 Seikagaku Kogyo Co Ltd ケラタン硫酸オリゴ糖画分の製造法
EP0795560A4 (fr) * 1994-12-01 1998-09-02 Seikagaku Kogyo Co Ltd Fraction d'oligosaccharide de sulfate de keratane et medicament la contenant
US5939403A (en) * 1994-12-01 1999-08-17 Seikagaku Corporation Keratan sulfate oligosaccharide fraction and pharmaceutical containing the same
US6159954A (en) * 1994-12-01 2000-12-12 Seikagaku Corporation Keratan sulfate oligosaccharide fraction and pharmaceutical containing the same
WO1997018222A2 (fr) * 1995-11-13 1997-05-22 Glycomed, Inc. Nouveaux glycosides oligosaccharidiques ayant des proprietes immunosuppressives et tolerogenes chez les mammiferes
WO1997018222A3 (fr) * 1995-11-13 1998-06-04 Glycomed Inc Nouveaux glycosides oligosaccharidiques ayant des proprietes immunosuppressives et tolerogenes chez les mammiferes
US5874411A (en) * 1995-11-13 1999-02-23 Glycomed Incorporated Oligosaccharide glycosides having mammalian immunosuppresive and tolerogenic properties
FR2751876A1 (fr) * 1996-07-31 1998-02-06 Roc Sa Utilisation d'un oligosaccharide comme immunomodulateur, composition dermato-cosmetique et methode de traitement cosmetique
WO1998004270A1 (fr) * 1996-07-31 1998-02-05 Roc Utilisation d'un oligosaccharide en tant qu'immunomodulateur dans une composition dermato-cosmetique
US6562954B1 (en) 1999-01-07 2003-05-13 Seikagaku Corporation Method for producing oligosaccharide, and novel oligosaccharide and pharmaceutical composition containing the same
US6436911B1 (en) 1999-07-21 2002-08-20 Seikagaku Corporation IL-12 production inhibitor
WO2005061523A1 (fr) * 2003-12-23 2005-07-07 Progen Industries Limited Composes mimetiques du glycosaminoglycane (gag)

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EP0643718A1 (fr) 1995-03-22
JPH07507319A (ja) 1995-08-10
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JP2823358B2 (ja) 1998-11-11
CA2118405A1 (fr) 1993-09-12

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