EP0591254A1 - Composes modifies immunosuppresseurs et tolerogenes de lewis?x et de lewis?a - Google Patents

Composes modifies immunosuppresseurs et tolerogenes de lewis?x et de lewis?a

Info

Publication number
EP0591254A1
EP0591254A1 EP92911318A EP92911318A EP0591254A1 EP 0591254 A1 EP0591254 A1 EP 0591254A1 EP 92911318 A EP92911318 A EP 92911318A EP 92911318 A EP92911318 A EP 92911318A EP 0591254 A1 EP0591254 A1 EP 0591254A1
Authority
EP
European Patent Office
Prior art keywords
compound
group
hydrogen
lewis
benzyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP92911318A
Other languages
German (de)
English (en)
Inventor
Robert M. Ippolito
Wasimul Haque
Cong Jiang
Rizk Hanna
Andre P. Venot
Pandurang V. Nikrad
Mohammed A. Kashem
Richard H. Smith
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glycomed Inc
Original Assignee
Alberta Research Council
Glycomed Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Alberta Research Council, Glycomed Inc filed Critical Alberta Research Council
Publication of EP0591254A1 publication Critical patent/EP0591254A1/fr
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention is directed to novel Lewis x and Lewis a 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 sialyl 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 .
  • selectins or LEC-CAM's
  • sialyl Lewis x and sialyl Lewis a and related compounds are difficult to chemically synthesize in high yield with anomeric specificity for the ⁇ (2 ⁇ 3) linkage of Neu5Ac to galactose.
  • ⁇ 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 x glycoside [ ⁇ Neu5Ac(2 ⁇ 3) ⁇ Gal(1 ⁇ 4) [ ⁇ Fuc(1 ⁇ 3)]- ⁇ GlcNAc-OR] or the sialyl Lewis a glycoside [i.e., ⁇ Neu5Ac(2 ⁇ 3) ⁇ Gal (1 ⁇ 3) [ ⁇ Fuc(1 ⁇ 4)] ⁇ GlcNAc-OR] where R is an aglycon of at least one carbon atom.
  • sialyl Lewis x sialyl Lewis a and related compounds can also be synthesized via
  • ⁇ Gal(1 ⁇ 4) ⁇ GlcNAc-OR the ⁇ Gal(1 ⁇ 3) ⁇ GlcNAc-OR, or the derivatized ⁇ Gal(1 ⁇ 3) ⁇ GlcNAc-OR backbone is first synthesized chemically and then the sialic acid residue (e.g., Neu5Ac) is attached to the galactose to form the ⁇ Neu5Ac(2 ⁇ 3) ⁇ Gal(1 ⁇ 3) ⁇ GlcNAc-OR or the ⁇ Neu5Ac(2 ⁇ 3) ⁇ Gal(1 ⁇ 4) ⁇ GlcNAc-OR structures by use of a compatible sialyltransferase and the fucose
  • the sialic acid residue e.g., Neu5Ac
  • the present invention is directed, in part, to the discovery that modified Lewis x and modified
  • the present invention is directed to compounds of Formula I:
  • R is selected from the group consisting of hydrogen, a saccharide-OR 14 , an oligosaccharide-OR 14 , or an aglycon having at least 1 carbon atom where R 14 is hydrogen or an aglycon of at least one carbon atom;
  • Y is selected from the group consisting of oxygen, sulfur, and -NH-;
  • R 1 is selected from the group consisting of hydrogen, -NH 2 , -N 3 , -NHSO 3 H, -NR 5 C(O)R 4 ,
  • -N C(R 5 ) 2 , -NHCH(R 5 ) 2 , -NHR 6 , -N(R 6 ) 2 , -OH, -OR 6 , -S(O)R 6 , -S(O) 2 R 6 and sulfate,
  • R 4 is selected from the group
  • alkyl of from 1 to 4 carbon atoms
  • R 7 is alkyl of from 1 to 4 carbon atoms, or alkyl of from 2 to 4 carbon atoms substituted with a hydroxyl group
  • R 8 and R 9 are independently selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms,
  • each R 5 is selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms
  • each R 6 is alkyl of from 1 to 4 carbon atoms
  • R 2 is selected from the group consisting of hydrogen, -N 3 , -NH 2 , -NHSO 3 H, -NR 11 C(O)R 10 ,
  • -N C(R 11 ) 2 , -NHCH(R 11 ) 2 , -NHR 12 , -N(R 12 ) 2 , -OH and -OR 12 ,
  • R 10 is selected from the group
  • alkyl of from 1 to 4 carbon atoms
  • R 13 is alkyl of from 1 to 4 carbon atoms, or alkyl of from 2 to 4 carbon atoms substituted with a hydroxyl group
  • R 14 and R 15 are independently selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms, each R 11 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, sulfate, and phosphate
  • X 1 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;
  • 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
  • R is selected from the group consisting of hydrogen, a saccharide-OR 14 , an oligosaccharide-OR 14 , or an aglycon having at least 1 carbon atom where R 14 is hydrogen or an aglycon of at least one carbon atom;
  • Y is selected from the group consisting of oxygen, sulfur, and -NH-;
  • R 1 is selected from the group consisting of hydrogen, -NH 2 , -N 3 , -NHSO 3 H, -NR 5 C(O)R 4 ,
  • -N C(R 5 ) 2 , -NHCH(R 5 ) 2 , -NHR 6 , -N(R 6 ) 2 , -OH, -OR 6 , -S(O) R6 , -S(O) 2 R 6 and sulfate,
  • R 4 is selected from the group
  • alkyl of from 1 to 4 carbon atoms
  • R 7 is alkyl of from 1 to 4 carbon atoms, or alkyl of from 2 to 4 carbon atoms substituted with a hydroxyl group
  • R 8 and R 9 are independently selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms,
  • each R 5 is selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms
  • each R 6 is alkyl of from 1 to 4 carbon atoms
  • R 10 is selected from the group
  • alkyl of from 1 to 4 carbon atoms
  • R 13 is alkyl of from 1 to 4 carbon atoms, or alkyl of from 2 to 4 carbon atoms substituted with a hydroxyl group
  • R 14 and R 15 are independently selected from the group consisting of hydrogen and alkyl of from 1 to 4 carbon atoms, each R 11 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, sulfate, and phosphate
  • X 1 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;
  • 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
  • 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
  • the present invention is directed to novel
  • intermediates useful in preparing the compounds of Formula I and Formula II are highly crystalline and can be produced in large quantities in high purity without the need for chromatography to separate these compounds.
  • the present invention is directed to a method for modulating a cell-mediated immune response to an antigen in a mammal which method comprises administering to said mammal an amount of a compound of Formula I or
  • the present invention is directed to a method for preparing the compounds of Formula I and II above and to the preparation of intermediates useful in preparing the compounds of Formula I and II.
  • Figure 1 illustrates reaction schemes for the synthesis of partially blocked N-acetyl glucosamine derivatives which are then used to prepare either modified Lewis x compounds or modified Lewis a
  • Figure 2 illustrates reaction schemes for the synthesis of blocked fucose derivatives which are then used to prepare either modified Lewis x or modified Lewis a compounds.
  • Figure 3 illustrates reaction schemes for the synthesis of partially blocked galactose derivatives which are then used to prepare either modified
  • FIG. 4 illustrates the synthesis of modified Lewis x compounds having a sulfate substituent in the 3 position of the galactose unit. In this scheme, the 2,3 positions of galactose are differentially blocked so that the 3-position can be selectively deblocked and then selectively converted to the sulfate substituent.
  • Figure 5 illustrates the synthesis of modified Lewis x compounds having a sulfate substituent in the 3 position of the galactose unit. In this scheme, the 2,3 positions of galactose are not
  • deprotection of the 3-position of the galactose unit also results in deprotection of the 2-position and subsequent reaction to form the sulfate at the 3-position does not proceed with 100% yield but rather some of the product has a sulfate substituent at the 2-position of the galactose which is then separated by
  • Figure 6 illustrates the synthesis of modified Lewis x derivatives bearing a sulfate substituent at the 3-position of the galactose and which utilize a 6-benzyl and 2-N-phthaloyl blocked glucosamine which can be later deblocked to provide for a glucosamine derivative.
  • FIG. 7 illustrates a second synthesis of modified Lewis x compounds bearing a sulfate
  • FIG. 9 illustrates the synthesis of the 6-azido derivative of GlcNAc-OR.
  • Figure 10 illustrates the synthesis of the 6-alkoxy derivatives and the 6-deoxy derivatives of GlcNAc.
  • Figure 11 illustrates the increase in footpad swelling of immunized mice arising from a DTH inflammatory response measured 24 hours after challenge with 10 ⁇ g of the L111 S-Layer protein antigen wherein some of the mice have been treated at 5 hours after the challenge with 100 ⁇ g of
  • Figure 12 illustrates the increase in footpad swelling of immunized mice arising from a DTH inflammatory response measured 24 hours after challenge with 20 ⁇ g of SuperCarrier (from Pierce,
  • Figure 13 demonstrates the effect that sulfated Lewis x -OR (SULFONATE-LeX) has on the induction of an immune response to an antigen where R is as defined in Figure 12.
  • Figure 14 illustrates the effect that sulfated Lewis x -OR (LeXsulf) and sialyl Lewis x -OR (SleX) on lung injury arising from the intranasal
  • Figure 15 demonstrates the long term
  • the present invention is directed, in part, to the discovery of novel Lewis x and novel Lewis a 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 mammal (e.g., a DTH response).
  • novel Lewis x and novel Lewis a 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 mammal (e.g., a DTH response).
  • cell-mediated immune response to an antigen 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
  • the cell-mediated immune response is a leucocyte-mediated response.
  • Lewis x (sometimes referred to "Le x ”) refers to the trisaccharide having the following structure:
  • the core ⁇ Gal(1 ⁇ 4) ⁇ GlcNAc structure of Lewis x is often referred as a "type II structure” or "LacNAc structure”.
  • Lewis a (sometimes referred to "Le a ”) refers to the trisaccharide having the following structure:
  • modified Lewis glycosides and derivatives thereof refer to derivatives of the Le x modified in one or more of the fucose, galactose and N-acetylglucosamine saccharide units of Lewis x 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 are different from glycoconjugates because such aglycon moieties are neither a protein nor a lipid capable of forming a micelle or other large
  • modified Lewis a glycosides and derivatives thereof refer to derivatives of the Le a modified in one or more of the fucose, galactose and N-acetylglucosamine saccharide units of Lewis a 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 are different from glycoconjugates because such aglycon moieties are neither a protein nor a lipid capable of forming a micelle or other large
  • the aglycon refers to non-saccharide containing residues having at least one carbon atom.
  • the aglycon 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 form -(CH 2 -CR 15 G) n - wherein n is an integer equal to 1 to 5;
  • R 15 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; and
  • Z is selected from the group consisting of hydrogen, methyl, phenyl, nitrophenyl and
  • each R 16 is independently alkyl of from 1 to 4 carbon atoms and R 17 is an alkenyl group of from 3 to 10 carbon atoms.
  • aglycons are known in the art.
  • the nitro group is reduced to an amino group which can be protected as N-trifluoroacetamido.
  • the trifluoroacetamido group is removed thereby unmasking the amino 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
  • Rana et al. 20 discloses a 6-trifluoroacetamidohexyl aglycon (-O-(CH 2 ) 6 -NHCOCF 3 ) in which the trifluoroacetamido protecting group can be removed unmasking the primary amino group.
  • allyl linking arms can be derivatized in the presence of 2-aminoethanethiol 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 14 or an oligosaccharide-OR 14 at the reducing sugar terminus (where R 14 is as defined above).
  • oligosaccharide refers to a
  • carbohydrate structure having from 2 to about 10 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-acetylgalactosamine, fucose, sialic acid, 3-deoxy-D,L-octulosonic acid, and the like.
  • 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
  • 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
  • such modification can result in the removal of an -OH functionality, the introduction of an amine functionality, the introduction of a halo
  • 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-O-methyl-Neu5Ac, 4-N- acetyl-Neu5Ac, 4,7-di-deoxy-Neu5Ac, 4-oxo-Neu5Ac, as well as the 6-thio analogues of Neu5Ac.
  • Gal ⁇ (1 ⁇ 3)GlcNAcO-disaccharide type I
  • the derivatized Gal ⁇ (1 ⁇ 4)GlcNAcO-disaccharide type II
  • enzymatic means for attaching L-fucose to the 3-position of the GlcNAc unit of ⁇ Gal(1 ⁇ 4) ⁇ GlcNAcO-lipid containing a sulfate on the 3-position of the galactose unit has been reported 28 .
  • the fucose employed, as its GDP-derivative is one which is compatible with the fucosyltransferase (e.g., ⁇ Gal(1 ⁇ 3/4) ⁇ GlcNAc ⁇ (1 ⁇ 3/4) fucosyltransferase).
  • the ⁇ Gal(1 ⁇ 3/4) ⁇ GlcNAc ⁇ (1 ⁇ 3/4) fucosyltransferase is readily isolated from human milk 13,14,15 . Additionally, it is contemplated that these fucose or fucosyl compounds will also be compatible with other fucosyltransferases of
  • any fucose compound which, as its GDP-derivative, is recognized by the ⁇ Gal(1 ⁇ 3/4)- ⁇ GlcNAc ⁇ (1 ⁇ 3/4) fucosyltransferase so as to bind to the enzyme and is then available for transfer to the compound of Formula III or IV:
  • the fucose unit has been chemically modified so as to introduce and/or remove one or more functionalities from this structure.
  • 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.
  • fucose Certain compatible analogues of fucose are known in the art and include, by way of example, 3-deoxy-fucose, arabinose, and the like. 16
  • guanosine 5'-( ⁇ -L-fucopyranosyl)diphosphate and any and all compatible salts thereof which has the formula:
  • GDP-fucose is preferably prepared by the method described by Jiang et al. 17 in U.S. Patent Application Serial No. 07/848,223 which is
  • compatible salts as it is used in relation to guanosine 5'-( ⁇ -L-fucopyranosyl)diphosphate refers to those salts of guanosine 5'- ( ⁇ -L-fucopyranosyl)diphosphate which readily form counter ions (i.e., cations) and which are
  • Suitable compatible salts include those prepared from counter ions such as sodium, potassium, lithium, calcium, magnesium, ammonium, mono-, di-, tri- or tetra-alkylammonium, iron, zinc, and the like.
  • pharmaceutically acceptable salts includes the pharmaceutically acceptable addition salts of the compounds of Formula I 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 -OX, -OX 1 , and -OX 2 refers to substituents which, with the oxygen of a hydroxyl group of the galactose unit and/or fucose group, form a sulfate group (i.e., -O-S(O) 2 -OH).
  • X, X 1 or X 2 is a sulfate
  • the resulting -OX, -OX 1 and/or -OX 2 group is -O-S(O) 2 -OH, which readily forms pharmaceutically acceptable salts thereof (e.g., -O-S(O) 2 -O-Na + ).
  • phosphate such as used to define the substituents -OX, -OX 1 , and -OX 2 refers to
  • substituents which, with the oxygen of a hydroxyl group of the galactose unit and/or fucose group, form a phosphate group i.e., -O-P(O) 2 -OH.
  • a phosphate group i.e., -O-P(O) 2 -OH.
  • the resulting -OX, -OX 1 and/or -OX 2 group is -O-P(O) 2 -OH, which readily forms pharmaceutically acceptable salts thereof (e.g., -O-P(O) 2 -O-Na + ).
  • removable blocking group refers to any group which when bound to one or more hydroxyl groups of the galactose, the N-acetylglucosamine, and/or the fucose units of Lewis x and Lewis a moieties prevents reactions from occurring at these hydroxyl groups and which protecting group can be removed by conventional chemical or enzymatic steps to
  • removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as benzyl, benzoyl acetyl, chloroacetyl, benzylidine,
  • t-butyldiphenylsilyl 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
  • One such additional contemplated blocking group is a ⁇ -galactose which can be removed
  • glycosyltransferases are employed to effect the sequential addition of one or more of sugar units onto a GlcNAc-OR saccharide structure, a derivatized GlcNAc-OR saccharide structure, a LacNAc-OR
  • Enzymatic means to prepare modified Lewis x compounds and modified Lewis a compounds can be used at different steps. For example, L-fucose can be enzymatically transferred onto the deblocked
  • LacNAc-OR structure (modified Lewis x compounds); or a ⁇ Gal(1 ⁇ 3) ⁇ GlcNAc-OR structure or a
  • the LacNAc-OR disaccharide can be made by an appropriate fucosyltransferase such as the ⁇ Gal(1 ⁇ 3/4) ⁇ GlcNAc ⁇ (1 ⁇ 3/4)fucosyltransferase which is readily obtained from human milk 13,14,15 .
  • the LacNAc-OR disaccharide can be made by an appropriate fucosyltransferase such as the ⁇ Gal(1 ⁇ 3/4) ⁇ GlcNAc ⁇ (1 ⁇ 3/4)fucosyltransferase which is readily obtained from human milk 13,14,15 .
  • the ⁇ Gal(1 ⁇ 3) ⁇ GlcNAc-OR disaccharide glycoside can be made chemically.
  • sulfotransferases may be used to effect sulfation at the 3-position of galactose on either the type I or type II structures. As is apparent, this can be followed by transfer of fucose using an appropriate fucosyltransferase as described above.
  • chemical and enzymatic means can be coupled wherein, for example, the sulfated, phosphorylated, or -CHR 18 COOH substituted LacNAc-OR structure or sulfated, phosphorylated, or -CHR 18 COOH substituted ⁇ Gal(1 ⁇ 3) ⁇ GlcNAc-OR structure is made chemically and the fucosyl group transferred
  • Chemical synthesis is a convenient method for preparing either the complete oligosaccharide glycoside; for chemically modifying a saccharide unit which can then be chemically or enzymatically coupled to an oligosaccharide glycoside; or for chemically preparing an oligosaccharide glycoside to which can be enzymatically coupled one or more saccharide units.
  • Lewis x and Lewis a and some analogues thereof are known in the art. These materials are generally assembled using suitably protected individual monosaccharides including the desirable glucosamine, fucose and galactose, lactose or ⁇ Gal(1 ⁇ 3) ⁇ GlcNAc
  • the chemical synthesis of all or part of the oligosaccharide glycosides 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.
  • 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.
  • a large variety of aglycon moieties are known in the art and can be attached with the proper configuration to the anomeric center of the reducing unit.
  • the aglycon is formed by
  • compound 5 can be blocked at the 3-hydroxyl group by reaction with, for example, allyl bromide and base (e.g., barium
  • 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.
  • compound 1 is converted to compound 12 (or the bromo analogue) by following similar procedures set forth above for compound 2.
  • Compound 16 is prepared by treating p-chlorothiophenol with 0.95 equivalents of potassium hydroxide in ethanol followed by heating the
  • Compound 18 is quickly deacetylated under Zemplen conditions (NaOMe, MeOH) to yield p- chlorophenyl ß-thiofucopyranoside 19 as a
  • temperatures of from about 15o to about 30oC and reaction times of about 1 to about 10 hours can be used.
  • step (b) maintaining the solution produced in step (a) above at about 50-55oC for a sufficient period of time so as to produce the 1,2,3,4-tetraacetylated derivative of L-fucose;
  • step (c) reacting the product produced in step (b) above with at least one equivalent of p-chlorothiophenol and from about 1 to about 3 equivalents of boron trifluoride etherate under conditions
  • step (e) contacting the p-chlorophenyl ß-thiofucopyranoside produced in step (d) with benzyl chloride or benzyl bromide in the presence of a base and under conditions sufficient to provide for p-chlorophenyl 2,3, 4-tri-O-benzyl-ß-thiofucopyranoside.
  • compound 23 can be converted to either a 3-acetyl (compound 24) or the 4-acetyl blocking group (not shown). In turn, both of these compounds are then blocked at the remaining hydroxyl group with a chloroacetyl blocking group by acetylation with chloroacetylchloride in pyridine/dichloromethane at about 0oC.
  • 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-O-benzylidene ß-D-thiogalactopyranoside, 29, is isolated and crystallized from hot isopropanol.
  • reaction conditions are not critical and preferably the reaction is conducted at from about 0"C to about 30oC and for about 1 to about 4 hours (preferably room temperature for 2 hours) to give crystalline benzyl 4,6-O-benzylidene 2-O-benzoyl-3-O-chloroacetyl-ß-D-thio-galactopyranoside, compound 31, in approximately 10-20% overall yield from galactose.
  • the advantage of this approach is that after subsequent assembly the blocked intermediates will be simply deblocked and modified by sulfation or phosphorylation.
  • the material is crystalline and the process obviates the need for chromatography.
  • the sulfates and phosphates of the galactose moiety of blocked Lewis a and Lewis x can also be made using compound 32 in the synthesis of these compound.
  • This compound is made by direct benzoylation of both the 2,3-hydroxyl groups of compound 29. However, after deblocking, both the 2 and 3 hydroxyl groups of galactose are then available for sulfation and phosphorylation and the selectivity is not asefficient.
  • 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 33 and 32a (shown in Figure 5) 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 50oC for about 1-2 hours.
  • 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-O-dibenzoyl-4-O-benzyl- ⁇ -D-thiogalactopyranoside (not shown).
  • this compound is chloroacetylated at the 6-hydroxyl group. After formation of the Lewis x or Lewis a structures, the chloroacetyl group can be
  • the present invention relates to a method for the preparation of benzyl 4,6-di-O-benzylidene-2-O- benzoyl-3-O-chloroacetyl-ß-D-thiogalactopyranoside which comprises the steps:
  • step (b) maintaining the solution produced in step (a) above at about 75-85oC for a sufficient period of time so as to produce the 1,2,3,4,6-pentaacetylated derivative of D-galactose;
  • dimethylformamide (DMF) solution containing the benzyl 4,6-di-O-benzylidene-ß-D-thiogalactopyranoside produced in step (e) above maintained at a temperature of from about -40oC to about -15oC for a sufficient period of time so as to provide benzyl 4,6-di-O-benzylidene-3-O-chloroacetyl- ⁇ -D- thiogalactopyranoside; and
  • Figure 4 illustrates one method for
  • the chloroacetyl protecting group at the 3-position of galactose is selectively removed and then converted to the sulfate, phosphate or -OCHR 18 COOH group. Also, as noted above, the
  • compound 7 and compound 33 are combined to form compound 37. This is accomplished by dissolving compound 7 and
  • the molecular sieves are removed by filtration by passing through celite and the recovered 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 of compound 37. This is then purified by conventional techniques such as column
  • compound 25 (or the 3-chloroacetyl analogue of compound 25 described above--not shown) can be used in place of compound 20 in the above synthesis. Removal of the
  • chloroacetyl blocking groups on the 3-hydroxyl of the galactose and the 4-hydroxyl of the fucose provides an facile route to the preparation of a disulfated, diphosphorylated Lewis x derivatives.
  • compound 40 can then be alkylated by first adding an appropriated base
  • benzyl bromide acetate (BrCH 2 COOBn) or other similar acetates (e.g.,
  • galactose which is purified and converted to its disodium salt by contacting a solution of this compound with a sodium form of Dowex 50 ⁇ 8.
  • FIG. 5 illustrates another method for
  • compound 46 is produced as a mixture of the 3-sulfate and the 2-sulfate (or 2,3-disulfate) which is separated by chromatography (e.g., column
  • lactose can be used in the methods of this invention in place LacNAc by merely a suitable blocking group at the 2-hydroxy of the glucose moiety of the lactose structure 49 .
  • Differential blocking of the lactose provides for a composition having a selectively removable blocking group at the 3 and/or 6 position of the galactose. This compound is then selectively deblocked at the 3 and/or 6 position and then derivatized to the 3 and/or 6 sulfate, phosphate or -OCHR 18 COOH.
  • FIG. 4 and 5 illustrate the synthesis of Lewis x structures
  • Lewis a structures are readily prepared in a similar manner, as illustrated in Figure 8, using appropriately blocked GlcNAc-OR structures.
  • the ⁇ Gal(1 ⁇ 3) ⁇ GlcNAc-OR structures can be prepared, for example, from compounds 5 and 35. Specifically, compound 35 is first converted to the 1- ⁇ -bromo derivative via known methodology (Norberg et al. 26 ) using bromine (Br 2 ) and tetraethylammonium bromide (Et 4 N + Br-) at about 0oC. About 1.5
  • compound 32 is converted to the 1- ⁇ -bromo derivative via known methodology (Norberg et al. 26 ) as described above and the resulting compound is then treated with sodium
  • both type I and type II structures can be made simultaneously by
  • dichloromethane containing molecular sieves to which is added about 1 equivalent (based on compound 15) of 2,6-di-t-butyl-4-methylpyridine.
  • the reaction is stirred for 30 minutes at room temperature and then cooled to -50oC.
  • An 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 -15oC over 2 hours and maintained at that temperature for an additional 5 hours. Afterwards, the solution is allowed to come to room temperature and stirred overnight.
  • the ratio of type I structure to type II structure resulting from this reaction can be improved by using the 2-NAc derivative of GlcNH 2 compound 15.
  • This compound can be readily prepared by reacting compound 15 with hydrazine, acetylating the resulting product with acetic anhydride/pyridine and then deacetylating the 3,4-hydroxyl groups by treatment with sodium methoxide/methanol.
  • the appropriately blocked type I [ ⁇ Gal(1 ⁇ 3) ⁇ GlcNAc-OR] and type II [ ⁇ Gal(l ⁇ 4) ⁇ GlcNAc-OR] structures can be selectively deblocked to provide for a hydroxyl group at the 3-position of galactose (or at the 6-position) and then sulfonated,
  • fucosyltransferase 29 The enzymatic transfer of fucose onto the 4-position of GlcNAc to form Lewis a and to the 3-position of GlcNAc to form Lewis x structures requires the prior synthesis of its nucleotide (GDP) derivatives. Synthesis of GDP- fucose is preferably accomplished in the manner recited by Jiang et al. 17 and which is exemplified in the examples hereunder.
  • GDP-fucose (GDP-Fuc) is then combined with the derivatized ⁇ Gal(1 ⁇ 4) ⁇ GlcNAc-OR compound or the derivatized ⁇ Gal(1 ⁇ 3) ⁇ GlcNAc-OR compound in the presence of a suitable fucosyltransferase (e.g., ⁇ Gal(1 ⁇ 3/4) ⁇ GlcNAc ⁇ (1 ⁇ 3/4) fucosyltransferase) under conditions wherein fucose is transferred to the 3 position of GlcNAc of the derivatized
  • a suitable fucosyltransferase e.g., ⁇ Gal(1 ⁇ 3/4) ⁇ GlcNAc ⁇ (1 ⁇ 3/4) fucosyltransferase
  • Suitable fucosylations conditions include the addition of the fucosyltransferase to a mixture of the derivatized ⁇ Gal(1 ⁇ 4) ⁇ GlcNAc-OR (or alternatively the derivatized ⁇ Gal(1 ⁇ 3) ⁇ GlcNAc-OR compound) and the GDP-fucose in a appropriate buffer such as 50 mM sodium cacodylate in
  • FIGS 6 and 7 illustrate two different syntheses for the retention of the 2-amino
  • 1- ⁇ -bromo-2,3,4,6-tetracetyl-galactose is prepared by first forming the peracetylated derivative of galactose, compound 26. Compound 26 is then converted to the 1- ⁇ -bromo derivative via known methodology
  • Compound 20 is then reacted with one equivalent of bromine in dichloromethane at -20oC for about 1 hour to provide for the 1- ⁇ -bromo derivative of compound 20.
  • the solution is then quenched with a cold aqueous sodium bicarbonate solution.
  • the organic solution is dried and concentrated to approximately half the original volume in vacuo at room temperature.
  • About 2 equivalents of this compound are then add to a dichloromethane solution of compound 48 that contains about 2 equivalents of mercuric bromide (HgBr 2 ), molecular sieves and tetraethylammonium bromide.
  • Compound 53 can then be sulfated in the same manner as described above for compound 45.
  • compound 53 can be differentially blocked at the 2,3 hydroxyl groups of the galactose in the same set forth above for compounds 29 - 31 so as to provide for compounds 54 and 55.
  • compound 55 is selectively deblocked with thiourea to provide for compound 56 in the same manner described above for compound 39 (to provide compound 40).
  • Compound 56 is then selectively sulfated in the manner described above to provide for compound 57.
  • compound 56 can be converted to the 3-phosphate group on the galactose by reaction with diphenylphosphorochloridate and 4-dimethyl-aminopyridine (1:1) in pyridine at 0oC.
  • Compound 56 can also be converted to the -CHR 18 COOH in the manner described above.
  • compound 60 which is a Lewis x analogue having a 2-amino glucose saccharide unit instead of a GlcNAc saccharide and further having a sulfate at the 3-position of the galactose saccharide unit.
  • 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 -20oC to 20oC to provide for compound 67.
  • the benzylidine group of compound 67 is then removed with 80% aqueous acetic acid at about 80oC for about 1-4 hours to provide for compound 68.
  • This compound is then selectively acetylated at the 6-position by use of approximately equimolar amounts of acetyl
  • the benzyl protecting group is then removed by hydrogenolysis (H 2 /Pd on C) to provide for compound 71.
  • Compound 71 in turn, is fucosylated in the same manner as described above for compound 48 (to provide for compound 49 as illustrated in Figure 6) so as to provide for compound 72.
  • Compound 72 is deacetylated by conventional techniques described above to provide for compound 73.
  • Compound 73 is then converted to compound 74 by conventional methodology (e.g., benzaldehyde dimethylacetal, DMF, pTSA), followed by selective acetylation at the 6-position of the partially protected GlcNH 2
  • the free hydroxyl groups of compound 74 can be acetylated with acetyl chloride/pyridine in the manner described above and the benzylidine group selectively opened by sodium cyanoborohydride and eerie or aluminum chloride to give the 2,3-diacetyl-4-benzyl-6-hydroxy derivative on the galactose moiety (not shown).
  • This compound is then functionalized at the 6-position of the galactose so as to contain a sulfate, phosphate or -CHR 18 COOH group at this position.
  • the 2,6 positions of the GlcNAc unit can be modified prior to coupling so as to provide for type I and type II structures modified at these positions which are then further modified in the manner described above to prepare the sulfated, phosphorylated or -CHR 18 COOH
  • the azido group is reduced to an amino group which can be protected as N-trifluoroacetamido.
  • 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.
  • the carboxylic acid, anhydride, chloride, or activated acid is selected so as to provide for an R 10 group (i.e., as part of the -NR 11 C(O)R 10 substituent) which is hydrogen or alkyl of from 1 to 4 carbon atoms,
  • an aldehyde or ketone of from 1 to 4 carbon atoms
  • 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(O)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,
  • the chloroformate has an R 13 group which is alkyl of from 1 to 4 carbon atoms,
  • the 1,2-ortho ester of the resulting compound is then opened by conventional techniques to provide a protected glycosyl donor such as the 1 ⁇ -bromo-2-acetyl-3,4,6-tribenzyl derivative of glucose.
  • This 1 ⁇ -bromo derivative is then converted to the glycoside (-OR) by
  • This compound is, in turn, converted to the 3,4-dibenzyl-6-deoxy-GlcNAc-OR by reaction with (C 4 H 9 ) 3 SnH in the presence of AIBN (azo bisisobutyronitrile) at 110oC followed by treatment with methanol/sodium methoxide.
  • AIBN azo bisisobutyronitrile
  • This compound can then be deprotected by conventional techniques to provide for the 6-deoxyGlcNAc-OR 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 9.
  • GlcNAc-OR compound 140
  • compound 140 is converted to the p-methoxybenzylidine blocked compound 141 by reaction with (CH 3 O) 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 O-C 6 H 4 -CH 2 Br to provide for compound 142 where X' is 4-CH 3 O-C 6 H 4 -CH 2 -.
  • Compound 142 is partially deprotected at the 4 and 6
  • the 6-mesylate, compound 144 is prepared by
  • compound 145 is then formed by reaction with sodium azide in dimethylformamide (DMF) and removal of the 3-blocking group with dichlorodicyanoquinone (DDQ) yields compound 146.
  • DMF dimethylformamide
  • DDQ dichlorodicyanoquinone
  • the 6-mesyl compound 144 can also be
  • the 6-azido compound 145 can be derivatized to the 6-amino at an appropriate point in the synthesis of the Lewis a or Lewisx analogues in the manner described above.
  • the -NH 2 group can be reacted, using conventional techniques, with:
  • 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(O)NH- substituent where alkylene is from 2 to 4 carbon atoms as reported by Wollenberg et al. 39 , U.S. Patent No.
  • the chloroformate has an R 7 group which is alkyl of from 1 to 4 carbon atoms,
  • the 6-alkoxy derivatives of GlcNAc can be prepared in the manner described in Figure 10.
  • compound 147 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 148.
  • Deprotection at the 4,6 positions by contacting compound 148 with acetic acid and water at about 80o-90oC provides for compound 149. Reaction of compound 149 with
  • dibutyltin oxide [(Bu) 2 SnO] and R 6 Br provides for the 6-alkoxy compound 150.
  • Conventional deprotection of the benzyl group with hydrogen in palladium/carbon yields compound 151.
  • compound 147 can be reacted with [C 6 H 5 C(O)] 2 O in pyridine to provide for a benzoyl protecting group (Bz) at the 3-position, i.e., compound 152.
  • Reaction of compound 152 with N-bromosuccinimide in carbon tetrachloride yields the 6-bromo compound 153.
  • Compound 153 can be reacted with tributyltin hydride [(Bu) 3 SnH] in toluene to provide for the 6-deoxy compound 155 which after conventional deprotection of the benzoyl groups with sodium methoxide in methanol gives the 6-deoxy compound 156.
  • the 6-SR 6 compounds are prepared from the 6mesyl derivative, compound 144, by reaction with potassium thioacetate, CH 3 C(O)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(O)R 6 and -S(O) 2 R 6 where
  • R 6 is alkyl of from 1 to 4 carbon atoms.
  • glycosides disclosed herein affect the cell mediated immune response in a number of ways. Specifically, these compounds can inhibit the ability of the immune response to become educated about a specific antigen when the compound is administered
  • the modified Lewis x and Lewis a glycosides disclosed herein can inhibit the effector phase of a cell-mediated immune
  • the modified Lewis x and Lewis a when administered after second or later exposures of the immune system to the same antigen.
  • glycosides disclosed herein can induce tolerance to antigens when administered at the time of second or later exposures of the immune system to the antigen.
  • the suppression of the inflammatory component of the immune response by the modified Lewis x and Lewis a glycosides disclosed herein is believed to require the initiation of a secondary immune response (i.e., a response to a second exposure to antigen).
  • the modified Lewis x and Lewis a glycoside is generally administered to the patient at least about 0.5 hours after an inflammatory episode, preferably, at least about 1 hour after, and most preferably, at least about 5 hours after an
  • the modified Lewis x and Lewis a glycosides disclosed herein are effective in suppressing cell-mediated immune responses to an antigen (eg. the inflammatory component of a DTH response) 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 to about 5 mg/kg of body weight.
  • an antigen eg. the inflammatory component of a DTH response
  • the specific dose employed is regulated by the
  • the modified Lewis x or Lewis a analogue is generally administered parenterally, such as intranasally, intrapulmonarily, transdermally and intravenously, although other forms of administration are
  • the suppression of a cell-mediated immune response eg. the inflammatory component of a DTH response
  • the suppression of a cell-mediated immune response is reduced by at least about 10% as opposed to control measured 24 hours after administration of the challenge to the mammal and 19 hours after administration of the modified Lewis x or Lewis a glycoside as per this invention.
  • administration of the modified Lewis x and Lewis a glycosides disclosed herein also imparts a tolerance to additional challenges from the same antigen. In this regard, re-challenge by the same antigen weeks after
  • the term "reducing sensitization” means that the modified Lewis x or Lewis a glycoside, 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 sensitized to the antigen administered at the same time as the modified Lewis x or Lewis a glycoside compound.
  • An "effective amount" of this compound is that amount which will reduce sensitization
  • the reduction in sensitization will be at least about 20% and more preferably at least about 30% or more.
  • modified Lewis x and Lewis a are particularly preferred.
  • glycoside compounds related to blood group are glycoside compounds related to blood group
  • determinants 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.
  • the specific dose employed is
  • “Simultaneous" administration 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.
  • the methods of this invention are generally achieved by use of a pharmaceutical composition suitable for use in the parenteral administration of an effective amount of an oligosaccharide glycoside related to a blood group determinant.
  • compositions comprise a pharmaceutically inert carrier such as water, buffered saline, etc. and an effective amount of a modified Lewis x or Lewis a glycoside compound so as to provide the above-noted dosage of the oligosaccharide glycoside when
  • compositions can be administered to a patient. It is contemplated that suitable pharmaceutical compositions can be
  • compositions for transdermal compositions or bandages etc., which are well known in the art.
  • BSA bovine serum albumin
  • DTH delayed-type hypersensitivity
  • IR-120 resin amberlite resin available from Rohm & Haas, Philadelphia, PA
  • Millipore Millipore Corp., Bedford, MA.
  • the first part relates to the synthetic procedures to make the recited compounds whereas the second part (part II) relates to the biological results.
  • Examples 1-24 illustrate syntheses of the described compounds.
  • reaction mixture is drained into a clean 20 L polyethylene pail.
  • the 50 L reactor is charged with 15 L of saturated sodium 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
  • the benzyl 2,3,4,6-tetra-O-acetyl-ß-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 dissolve the material. The solution is 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-ß-D-thiogalactopyranoside with 80:20 ethyl acetate:
  • isopropanol is added and the mixture is heated to near reflux. Most of the product goes into the hot isopropanol after warming for several hours. The mixture is cooled and ice added to the bath and cooling continued overnight to give a precipitate. After filtering and drying the precipitate, 760 g of benzyl-4,6-O-benzylidene-ß-D-thiogalactopyranoside is obtained.
  • the separated organic layer is then dried over sodium sulphate and evaporated to dryness.
  • the residue is taken up in 1 L of dry methanol in a 2 L round bottom flask and treated with 1 g of freshly surfaced sodium. The reaction is kept under
  • p-Chlorophenyl-ß-L-thiofucopyranoside is dissolved in 7 L of dry dimethylsulphoxide.
  • 600 g of powdered KOH is added dropwise to the stirring solution and the mixture stirred overnight at room temperature.
  • T.l.c. indicates incomplete reaction so an additional 300 g of powdered KOH is added to the reaction mixture followed 30 minutes later by 425 mL of benzyl chloride.
  • the solution is stirred at room temperature until t.l.c. indicates the reaction is complete. If the reaction is not complete after 24 hours, powdered KOH is added followed by 200 mL of benzyl chloride.
  • a 20L glass reactor was charged with 8 L of dichloroethane, 1 L of acetic anhydride and 1 kg of anhydrous sodium acetate. To the stirring 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 solution over 3-4 hours and the solution maintained at reflux for 36 hours. During the last hour of reflux 200 mL of water was added dropwise to the solution. The reaction was then cooled and added to 35 L of ice water in a 50 L stirred reactor. The organic layer was removed and then water washed a second time with an additional 20 L of water.
  • reaction completion by t.l.c. the reaction mixture was filtered through a buchner funnel of silica and the organic layer washed twice with water, twice with a 5% solution of potassium iodide and twice with a saturated solution of sodium bicarbonate.
  • dichloromethane were added to the mixture and was filtered over celite, filtrate was washed with saturated aqueous sodium hydrogen carbonate (200 mL) and then with water (200 mL), aqueous hydrogen chloride (0.5N, 200 mL) and water (200 mL),
  • Glucosamine hydrochloride 100 g, 0.46 mol was added to a solution of sodium methoxide in methanol which was prepared from equimolar amount of sodium metal in methanol (0.5 L). The resultant mixture was treated with equimolar equivalent of phthalic anhydride and triethylamine (80 mL). The mixture was then stirred for 2 hours, filtered and the solid was dried in vacuum for 12 hours. The dry solid was dissolved in pyridine (300 mL) and treated with acetic anhydride (200 mL, 2.1 mol). The mixture was then stirred at room temperature for 48 hours.
  • 2-deoxy-2-phthalamido-1,3,4,6-tetra-O-acetyl- ⁇ -D-glucopyranoside 1 (20g, 41.9mmol) was treated with hydrogen bromide solution in acetic acid (30%, 200mL) and stirred at room temperature for 2 hrs. The mixture was then poured into an ice water mixture and extracted with dicloromethane.
  • Ethyl 2-deoxy-2-phthalamido-3,4,6-tri-O-acetyl- ⁇ -D-glucopyranosyide (compound 13) from example 17 was taken up in 100 mL of dry methanol and treated with 100 mg of sodium metal. The solution was stirred at room temperature for 24 hours and then neutralized with Amberlite [R-120(H+)] resin, filtered, and evaporated to dryness in vacuo. This compound was used in the preparation of compound 15 and compound 66.
  • tetrabutylammonium bromide (0.983 g, 3.05 mmol). The mixture was heated at 150oC 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) was added and the reaction heated to 110oC for 36 hours.
  • fucosylation of the sulfated Lewis x and Lewis a structures can be achieved by use of an appropriate fucosyltransferase which are well known in the art.
  • Enzymatic fucosylation requires the use of GDP-fucose. Accordingly, the purpose of this example is to illustrate the synthesis of GDP-fucose. This is achieved in a 3 step process as illustrated below:
  • Tetra-n-butylammonium hydroxide (40% aq. w/w, about 150g) was added dropwise to a solution of phosphoric acid (85% aq, w/w, 18g, 0.155 mmol) in water (150 mL) until the pH reached 7. Water was then evaporated in vacuo to give a syrup which was co-evaporated with dry aceto-nitrile (2 ⁇ 400 mL) followed by dry toluene (2 ⁇ 400 mL). The resulting white solid (75g) was dried in vacuo and stored over phosphorus pentoxide under vacuum until used.
  • Guanosine 5'-( ⁇ -1-fucopyranosyl)-diphosphate was prepared from ⁇ -L-fucopyranosyl-1-phosphate using two different art recognized procedures as set forth below:
  • ⁇ -L-fucopyranosyl-1-phosphate and guanosine 5'-mono-phosphomorpholidate (4-morpholine-N,N'-di-cyclohexyl-carboxamidine salt, available from Sigma, St. Louis, Missouri, "GMP-morpholidate") were reacted as described in a recent modification 54,56 of Nunez's original procedure 55 .
  • tri-n-octylamine (0.800g, available from Aldrich Chemical Company, Milwaukee, Wisconsin) was added to a mixture of ⁇ -L-fucopyranosyl-1-phosphate (triethylammonium salt, 1.00g, about 2.20 mmol) in dry pyridine (10 mL) under nitrogen the solvent removed in vacuo. The process was repeated three times with care to allow only dry. air to enter the flask.
  • GMP morpholidate (2.4g, about 3.30 mmol) was dissolved in a 1:1 mixture of dry dimethylformamide and pyridine (10 mL). The solvents were evaporated in vacuo and the procedure repeated three times as above. The residue was dissolved in the same mixture of solvents (20 mL) and the solution added to the reaction flask accompanied by crushed
  • Fractions 29 to 45 and 47 to 57 were separately pooled and freeze-dried providing recovered ⁇ -L-fuco-pyranosyl-1-phosphate (0.264g and 0.223g, respectively, in which the second fraction contains some impurities). Occasionally, pooling of
  • guanosine 5'-( ⁇ -1-fucopyranosyl)-diphosphate in good purity ( 1 H-n.m.r.).
  • all the material enriched in guanosine 5'-( ⁇ -1-fuco-pyranosyl)-diphosphate was dissolved in a minimum amount of water and run on the same column which had been regenerated by washing with large amounts of
  • ⁇ -L-fucopyranosyl-1-phosphate and guanosine 5'-monophosphomorpholidate (4-morpholine-N,N'-di-cyclohexyl-carboxamidine salt -- "GMP-morpholidate") were reacted in dry pyridine as indicated in the original procedure 55 . Accordingly, the ⁇ -L- fucopyranosyl-1-phosphate (triethyl-ammonium salt, 0.528g, about 1.18 mmol) was dissolved in dry pyridine (20 mL) and the solvent removed in vacuo. The process was repeated three times with care to allow only dry air to enter the flask.
  • GMP- morpholidate (1.2g, 1.65 mmol) and pyridine (20 mL) were added into the reaction flask, the solvent evaporated in vacuo and the process repeated three times as above. Pyridine (20 mL) was added to the final residue and the heterogeneous mixture was stirred for 3 to 4 days at room temperature under nitrogen. An insoluble mass was formed which had to be occasionally broken down by sonication.
  • Examples 26-29 illustrate the immunomodulatory, antiinflammatory, and tolerogenic properties of compounds disclosed herein.
  • DTH inflammatory responses were measured using the mouse footpad swelling assay as described by Smith and Ziola 50 . Briefly, groups of Balb/c mice were immunized with S-layer protein, a bacterial surface protein 51 from Clostridium
  • mice containing a sulfate substituent at the 3-position of galactose (compound 47), groups of mice
  • LPS lipopolysaccharide
  • mice were sensitized with 5 ⁇ g/mouse of LPS in 50 ⁇ l of PBS intranasally under light anesthesia.
  • mice are anethesitized with Metofane (Pitman-Moore Ltd., Mississauga, Ontario, Canada) and a 50 ⁇ l drop of compound is placed on the nares of the mouse and is inhaled.
  • Metofane Pane-Moore Ltd., Mississauga, Ontario, Canada
  • mice Five hours later, 100 ⁇ g/mouse of sialyl Lewis x or sulfated Lewis x in 200 ⁇ l of PBS are given to the mouse intravenously. After 24 hours, the mice are sacrificed and the lungs removed and weighed. The weight of the lungs of mice treated with either sialyl Lewis x or sulfated Lewis x are compared against control (i.e., mice treated with LPS but to which neither sialyl Lewis x or Lewis x has been
  • FIG 13 which illustrates that sulfated Lewis x provides about 50% reduction whereas sialyl Lewis a provides only about a 30% reduction in the DTH inflammatory response in lungs. This suggests that not only do both sialyl Lewis x and sulfated Lewis x can be useful in reducing inflamation in lungs exposed to antigen, for example Acute Respiratory Distress Syndrome but that sulfated Lewis x actually provides significantly enhanced results.
  • Control groups were either not immunized or received 20 ⁇ L of phosphate-buffered saline (PBS) in place of either sialyl Lewis x or sulfated Lewis x .
  • PBS phosphate-buffered saline
  • the footpad swelling was measured 24 hours later with a Mitutoyo Engineering micrometer.
  • FIG. 14 shows that administering sialyl Lewis x or sulfated Lewis x to the mice at the time of immunization reduces the induction of an immune response to an antigen as compared to PBS control. This suggests that administration of a compound of this invention at the time of antigen immunization will reduce the ability of the mammal to become educated concerning this antigen.
  • Example 29 Persistence of Suppression of the DTH
  • mice treated with sialyl Lewis x and sulfated Lewis x in Example 26 were re-challenged 6 weeks after primayr immmunization with 20 ⁇ g/mouse with SC. Untreated controls responded with the usual degree of footpad swelling whereas all other groups showed reduced footpad swelling as shown in Figure 15.
  • Lewis a 3- or 6-phosphated Lewis x , 3-6-phosphated Lewis a , and the following fucosed substituted materials: 3- or 4- sulfated or phosphated fucose groups on Lewis x or Lewis a and the like.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Epidemiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Saccharide Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Cette invention concerne de nouveaux analogues de Lewisx et de Lewisa, des compositions pharmaceutiques contenant ces analogues, ainsi que leurs procédés de préparation et d'utilisation.
EP92911318A 1991-06-10 1992-06-09 Composes modifies immunosuppresseurs et tolerogenes de lewis?x et de lewis?a Ceased EP0591254A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US71416191A 1991-06-10 1991-06-10
US714161 1991-06-10
US88901792A 1992-05-26 1992-05-26
PCT/CA1992/000245 WO1992022564A1 (fr) 1991-06-10 1992-06-09 Composes modifies immunosuppresseurs et tolerogenes de lewisx et de lewis?a¿
US889017 1997-07-07

Publications (1)

Publication Number Publication Date
EP0591254A1 true EP0591254A1 (fr) 1994-04-13

Family

ID=27109107

Family Applications (1)

Application Number Title Priority Date Filing Date
EP92911318A Ceased EP0591254A1 (fr) 1991-06-10 1992-06-09 Composes modifies immunosuppresseurs et tolerogenes de lewis?x et de lewis?a

Country Status (4)

Country Link
EP (1) EP0591254A1 (fr)
JP (1) JPH06510746A (fr)
CA (1) CA2110707A1 (fr)
WO (1) WO1992022564A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5559103A (en) * 1993-07-21 1996-09-24 Cytel Corporation Bivalent sialyl X saccharides
WO1996016071A1 (fr) * 1994-11-21 1996-05-30 Glycomed, Inc. PROCEDES POUR SYNTHETISER DE COMPOSES DE LEWISx SUBSTITUES EN 3'
JPH0952902A (ja) * 1995-08-09 1997-02-25 Daikin Ind Ltd フッ素含有シアリルルイスｘ誘導体及びその合成中間体
US5874411A (en) * 1995-11-13 1999-02-23 Glycomed Incorporated Oligosaccharide glycosides having mammalian immunosuppresive and tolerogenic properties
US5830871A (en) * 1996-10-28 1998-11-03 The Scripps Research Institute Inhibitors of E-, P- and L-selectin binding
CA2299295C (fr) * 1997-08-08 2010-11-30 Aventis Pharma Deutschland Gmbh Derives substitues du tetrahydropyrane, leur procede de production, leur utilisation comme medicaments ou agents diagnostiques, et medicaments les contenant
TWI330641B (en) 2002-12-24 2010-09-21 Yasuhiro Kajihara Sugar chain asparagine derivatives

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9222564A1 *

Also Published As

Publication number Publication date
JPH06510746A (ja) 1994-12-01
CA2110707A1 (fr) 1992-12-23
WO1992022564A1 (fr) 1992-12-23

Similar Documents

Publication Publication Date Title
US5580858A (en) Immunosuppressive and tolerogenic modified Lewisx compounds
US5646123A (en) Time dependent administration of oligosaccharide glycosides related to blood group determinants having a type I or type II core structure in reducing inflammation in a sensitized mammal arising form exposure to an antigen
US5550155A (en) Methods for the synthesis of monofucosylated oligosaccharides terminating in di-N-acetyllactosaminyl structures
EP0589933B1 (fr) Methodes de synthese enzymatique de glycosides d'oligosaccharides a sialylation alpha
US5939290A (en) Modified sialyl Lewisx compounds
US6013779A (en) Process for preparation of glycosides of tumor-associated carbohydrate antigens
US5874411A (en) Oligosaccharide glycosides having mammalian immunosuppresive and tolerogenic properties
US5872096A (en) Modified sialyl Lewisa compounds
EP0591254A1 (fr) Composes modifies immunosuppresseurs et tolerogenes de lewis?x et de lewis?a
WO1992022301A1 (fr) Derives d'oligosaccharide immunosuppresseurs et tolerogenes
WO1993024505A1 (fr) Reduction de l'inflammation par administration dependante dans le temps d'oligosaccharides glycosides relatifs a des determinants de groupes sanguins
JP2823358B2 (ja) 免疫抑制性および寛容原性修飾ルイス▲上ｃ▼およびＬａｃＮＡｃ化合物
US5220008A (en) Oligosaccharide inhibitors for influenza virus
US5254676A (en) Oligosaccharide inhibitors for influenza virus
WO1988000951A1 (fr) Procede de preparation d'oxazolines de peracetyle
WO1996040702A1 (fr) Analogues kojibiosides modifies
WO1996016071A1 (fr) PROCEDES POUR SYNTHETISER DE COMPOSES DE LEWISx SUBSTITUES EN 3'

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19940107

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU MC NL SE

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: GLYCOMED INCORPORATED

17Q First examination report despatched

Effective date: 19951110

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 19990121