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WO1997014707A1 - Analogues de sialyle lewisx utilises en tant qu'inhibiteurs de l'adhesion cellulaire - Google Patents

Analogues de sialyle lewisx utilises en tant qu'inhibiteurs de l'adhesion cellulaire Download PDF

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Publication number
WO1997014707A1
WO1997014707A1 PCT/US1996/016559 US9616559W WO9714707A1 WO 1997014707 A1 WO1997014707 A1 WO 1997014707A1 US 9616559 W US9616559 W US 9616559W WO 9714707 A1 WO9714707 A1 WO 9714707A1
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group
lipid
compound
alkyl
hydrogen
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PCT/US1996/016559
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Shawn A. Defrees
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Cytel Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • 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 relates to compounds that inhibit cellular adhesion, and more particularly relates to analogue compounds of sialyl Lewis x (sialyl Le x or SLe x ) that inhibit selectin-mediated cellular adhesion, compositions containing and processes for using the same, and processes for preparing those analogues.
  • sialyl Lewis x sialyl Le x or SLe x
  • Vascular endothelial cells and blood platelets play key roles in a number of biological responses by selectively binding certain cells, for instance phagocytic leukocytes, in the bloodstream.
  • endothelial cells preferentially bind monocytes and granulocytes prior to their migration through the blood vessel wall and into surrounding tissue in an inflammatory response.
  • Certain inflammation-triggering compounds are known to act directly on the vascular endothelium to promote the adhesion of leukocytes to vessel walls. Cells then move through the walls and into areas of injury or infection.
  • Circulating cancer cells apparently take advantage of the body's normal inflammatory mechanisms and bind to areas of blood vessel walls where the endothelium is activated.
  • Blood platelets are also involved in similar responses. Platelets are known to become activated during the initiation of hemostasis and undergo major morphological, biochemical, and functional changes (e.g., rapid granule exocytosis, or degranulation), in which the platelet alpha granule membrane fuses with the external plasma membrane. As a result, new cell surface proteins become expressed that confer on the activated platelet new functions, such as the ability to bind both other activated platelets and other cells.
  • Activated platelets are recruited into growing thrombi, or are cleared rapidly from the blood circulation. Activated platelets are known to bind to phagocytic leukocytes, including monocytes and neutrophils. Examples of pathological and other biological processes that are thought to be mediated by this process include atherosclerosis, blood clotting and inflammation.
  • E-selectin endothelial leukocyte adhesion molecule-1
  • P-selectin granule membrane protein-140
  • E-selectin has been shown to mediate
  • E-selectin binds human neutrophils, monocytes, eosinophils, certain
  • T-lymphocytes [Graber et al., J. Immunol., 145:819 (1990)], NK cells, and the promyelocytic cell line HL-60.
  • E-selectin is inducibly expressed on vascular endothelial cells [Bevilacqua et al., Science, 243: 1160-1165 (1989) and Hession et al., Proc. Natl. Acad. Sci.,
  • IL-I ⁇ interleukin I ⁇
  • tumor necrosis factor ⁇ tumor necrosis factor ⁇
  • TNF ⁇ bacterial endotoxin
  • bacterial endotoxin lipopolysaccharide
  • P-selectin also known as GMP-140 and PADGEM
  • GMP-140 and PADGEM P-selectin
  • PADGEM protein-derived neurotrophic factor
  • activated platelets that express P-selectin on their surface are known to bind to monocytes and neutrophils [Jungi et al., Blood, 67:629-636 (1986)], and also to bind monocyte-like cell lines, e.g., HL-60 and U937 [Jungi et al., Blood, 67:629-636 (1986); Silverstein et al., J. Clin. Invest., 79:867-874 (1987)].
  • P-selectin is an alpha granule membrane protein of molecular mass 140,000 that is expressed on the surface of activated platelets upon platelet stimulation and granule secretion [Hsu-Lin et al., J. Clin. Chem., 259:9121-9126 (1984); Stenberg et al., J. Cell Biol., 101:880-886 (1985); Berman et al., J. Clin. Invest., 78: 130-137 (1986)].
  • a third receptor is the lymphocyte homing receptor, MEL- 14 antigen or its human counterpart LAM-1 (L-selectin) [Gallatin et al., Nature, 304:30-34 (1983); Siegellman et al., Science, 243: 1165-1172 (1989); Rosen, Cell Biology, 1:913-919 (1989); and Lasky et al., Cell, 56: 1045-1055 (1989)].
  • LAM-1 L-selectin
  • selectin has been suggested for a general class of receptors, which includes E-selectin (ELAM-1), P-selectin (GMP-140) and L-selectin (MEL-14), because of their lectin-like domain and the selective nature of their adhesive functions.
  • E-selectin E-selectin
  • GMP-140 P-selectin
  • MEL-14 L-selectin
  • the structure and function of selectin receptors has been elucidated by cloning and expression of full length cDNA encoding each of the above receptors [Bevilacqua et al., Science, 243:1160-1165 (1989), (ELAM-1); Geng et al., Nature. 343:757-760 (1990), (GMP-140); and Lasky et al., Cell, 56: 1045-1055 (1989), (MEL-14 antigen)].
  • the extracellular portion of selectins can be divided into three segments based on homologies to previously described proteins.
  • the N-terminal region (about 120 amino acids) is related to the C-type mammalian lectin protein family as described by Drickamer, J. Biol. Chem., 263:9557-9560 (1988) that induces low affinity IgE receptor CD23.
  • a polypeptide segment follows, which has a sequence that is related to proteins containing the epidermal growth factor (EGF) motif.
  • EGF epidermal growth factor
  • U.S. Patent No. 5,079,353 and its divisional Patent No. 5,296,594 teach the synthesis and use of the sialyl Le x and sialyl Le a antigens that are present in cancerous tissues, and are ligands for the before-described selectin receptors.
  • U.S. Patent No. 5,079,353 and its divisional Patent No. 5,296,594 teach the synthesis and use of the sialyl Le x and sialyl Le a antigens that are present in cancerous tissues, and are ligands for the before-described selectin receptors.
  • 5,143,712 teaches the binding interactions between various receptors such as ELAM-1 (E-selectin) and ligands such as sialyl Le x as well as ligands containing a plurality of N-acetyllactosamine (LacNAc) units along with a terminal sialyl group and one or more fucosyl groups that are bonded to the GlcNAc portion of a LacNAc unit.
  • ELAM-1 E-selectin
  • ligands such as sialyl Le x
  • LacNAc N-acetyllactosamine
  • the present invention contemplates a sialyl Le x (SLe x ) analogue compound that inhibits the adhesion of cells that express SLe x on their surfaces to a selectin receptor, intermediate compounds in the synthesis of an inhibitor, as well as a process for preparing such intermediates and a pharmaceutical composition containing an inhibitor.
  • SLe x sialyl Le x
  • compositions comprising a pharmaceutically acceptable diluent having dissolved or dispersed therein a cellular adhesion-inhibiting amount of a compound of the formula
  • Z is selected from the group consisting of hydrogen, C 1 -C 6 acyl and ,
  • R 1 is selected from the group consisting of a linking group for a lipid, a lipid, a linking group with attached lipid, an aryl, a substituted aryl and a phenyl C 1 -C 3 alkylene group, wherein an aryl group has one five-membered aromatic ring, one six-membered aromatic ring or two fused six-membered aromatic rings, which rings are selected from the group consisting of hydrocarbyl, monooxahydrocarbyl,
  • a substituted aryl group is said aryl group having a substituent selected from the group consisting of a halo, trifluoromethyl, nitro, C 1 -C 12 alkyl, C 1 -C 12 alkoxy, amino, mono-C 1 -C 12
  • R 1 Y is allyloxycarbonyl or
  • R 2 is selected from the group consisting of hydrogen, a linking group for a lipid, a lipid, a linking group with attached lipid, C 1 -C 18 straight chain, branched chain or cyclic hydrocarbyl, C 1 -C 6 alkyl C 1 -C 5 alkylene ⁇ -carboxylate, ⁇ -tri(C 1 -C 4
  • R 4 is an alkyl group;
  • R 5 is selected from the group consisting of hydrogen, benzyl,
  • R 4 is a member selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, benzyl, pentyl and hexyl.
  • the present invention further provides intermediates which are useful in the preparation of the pharmaceutical compositions above.
  • the present invention further provides intermediates which are useful in the preparation of the pharmaceutical compositions above.
  • the present invention further provides intermediates which are useful in the preparation of the pharmaceutical compositions above.
  • intermediates are either lactones or esters, having the formulae (Va), (Vb) and (VI), respectively,
  • the invention further provides a process for the preparation of compounds of Formula IV, comprising:
  • compositions of the present invention are useful in methods of inhibiting intercellular adhesion in a patient for a disease process, such as inflammation.
  • the selectin receptor such as E-Selectin or P-Selectin, may be expressed on vascular endothelial cells or platelets.
  • the inflammatory process may be, for example, septic shock, wound associated sepsis, rheumatoid arthritis, post-ischemic leukocyte-mediated tissue damage (reperfusion injury), frost-bite injury or shock, acute leukocyte-mediated lung injury (e.g.
  • atopic dermatitis psoriasis
  • inflammatory bowel disease Various platelet-mediated pathologies such as atherosclerosis and clotting can also be treated.
  • tumor metastasis can be inhibited or prevented by inhibiting the adhesion of circulating cancer cells. Examples include carcinoma of the colon and melanoma.
  • Figure 1 shows a reaction scheme for the preparation of compound 62.
  • Figure 2 shows a reaction scheme for the preparation of compound 73.
  • Figure 3 shows a reaction scheme for the preparation of compound 78.
  • Figure 4 shows a reaction scheme for the preparation of compound 85.
  • Figure 5 shows a reaction scheme for the preparation of compound 100.
  • Figure 6 shows a reaction scheme for the preparation of compound 115.
  • Figure 7 shows a reaction scheme for the preparation of compounds 116 to 120.
  • oligosaccharide moieties of the present invention follows the conventional nomenclature. Standard abbreviations for individual monosaccharides are used. For instance, 2-N-acetylglucosamine is represented by GlcNAc, 2-N-acetylgalactosamine is GalNAc, fucose is Fuc, fructose is Fru, galactose in Gal, glucose is Glc, and mannose is Man. Unless otherwise indicated, all sugars except fucose (L-isomer) are D-isomers in the cyclic configuration (e.g. , pyranose or furanose). The two anomers of the cyclic forms are represented by ⁇ and ⁇ .
  • the monosaccharides are generally linked by glycosidic bonds to form oligo- and polysaccharides.
  • the orientation of the bond with respect to the plane of the rings is indicated by ⁇ and ⁇ .
  • the particular carbon atoms that form the bond between the two monosaccharides are also noted.
  • a ⁇ glycosidic bond between C-1 of galactose and C-4 of glucose is represented by Gal ⁇ 1 ⁇ 4Glc.
  • the designation a means the hydroxyl attached to C-1 (C-2 in NeuAc) is below the plane of the ring and ⁇ is above the ring.
  • the a designation means the hydroxyl is above the ring and ⁇ means it is below.
  • the present invention contemplates a SLe x analogue compound of structural Formula A, below, which structural formula encompasses a pentasaccharide compound of Formula I that is an analogue of sialyl Le x , as well as its penta- and tetrasaccharide precursors of Formulas H and III, respectively.
  • a compound of structural Formula I inhibits cellular adhesion mediated by a selectin cell surface receptor.
  • Z is hydrogen (H) or C 1 -C 6 acyl, in which case a compound of Formula III is defined, or an ⁇ -L-fucosyl whose hydroxyl groups are free or blocked with a protecting group (benzyl or C 1 -C 6 acyl) thereby defining a compound of Formula I or II, depending upon the identities of R 3 , R 4 and R 5 (R 3-5 ) groups;
  • Y is selected from the group consisting of C(O), SO 2 , HNC(O), OC(O) and SC(O);
  • R 1 is selected from the group consisting of a linking group for a lipid, a lipid, a linking group with attached lipid, an aryl, a substituted aryl and a phenyl C 1 -C 3 alkylene group, wherein an aryl group has one five- or six-membered aromatic ring, fused five/six-membered aromatic rings, or two fused six-membered aromatic rings, which rings are selected from the group consisting of hydrocarbyl, monooxahydrocarbyl, monothiahydrocarbyl, monoazahydrocarbyl and diazahydrocarbyl rings, and a substituted aryl group is a before-mentioned aryl group having a substituent selected from the group consisting of a halo, trifluoromethyl, nitro, C 1 -C 18 alkyl, C 1 -C 18 alkoxy, amino, mono- C 1 -C 18 alkylamino, di-C 1 -C 18
  • R 1 Y is allyloxycarbonyl or chloroacetyl
  • R 2 is selected from the group consisting of hydrogen, a linking group for a lipid, a lipid, a linking group with attached lipid, C 1 -C 18 straight chain, branched chain or cyclic hydrocarbyl, C 1 -C 6 alkyl C 1 -C 5 alkylene ⁇ -carboxylate, ⁇ -tri(C 1 -C 4
  • R 3 is hydrogen or C 1 -C 6 acyl
  • R 4 is hydrogen, C 1 -C 6 alkyl or benzyl
  • R 5 is selected from the group consisting of hydrogen, benzyl, methoxybenzyl, dimethoxybenzyl and C 1 -C 6 acyl;
  • R 7 is methyl (CH 3 ) or hydroxymethyl (CH 2 OH);
  • X is selected from the group consisting of C 1 -C 6 acyloxy, C 2 -C 6 hydroxylacyloxy, hydroxy, halo and azido.
  • Y can be one of a number of groups.
  • Y is C(O)
  • R 1 Y is an acyl substituent group so that an amide is formed with the saccharide amine nitrogen atom.
  • R 1 Y forms a sulfonyl substituent group so that a sulfonamide is formed with the saccharide amine nitrogen atom.
  • Y is HNC(O)
  • R 1 Y forms an aminocarbonyl substituent group so that a urea substituent is formed with that saccharide nitrogen atom.
  • a urethane substituent is formed with the saccharide amine nitrogen where Y is oxycarbonyl, OC(O), whereas a thiourethane is formed where Y is thiocarbonyl, SC(O).
  • a Y group is preferably a carbonyl group [C(O)].
  • R 1 Y group can also be an allyloxycarbonyl or a chloroacetyl group.
  • An allyloxycarbonyl R 1 Y group is particularly preferred for a compound of Formula III as it provides a readily replaceable R 1 group.
  • An R 1 Y allyloxycarbonyl or chloroacetyl group is present only in a compound of Formula III, and is not present in a compound of any of Formulas I, II, A, B or C (Formulas B and C are shown hereinafter).
  • an R 1 group can be an aryl or substituted aryl group.
  • Contemplated aryl groups are those that contain one aromatic five- or six-membered ring, fused five- and six- (five/six-) membered rings or two fused aromatic six-membered rings and include hydrocarbyl groups such as phenyl and naphthyl, as well as
  • hydrocarbyl groups bearing an oxygen, a sulfur, or one or two nitrogen atoms that replace ring carbon atoms (mono- or diazahydrocarbyl).
  • exemplary aryl groups include furyl, thienyl, pyridyl, pyrazinyl, benzofuranyl (benzo[b]furyl), isobenzofuranyl
  • Each of those aryl groups can be unsubstituted, or each can have a substituent selected from the group consisting of halo, trifluoromethyl, nitro, C 1 -C 18 alkyl, C 1 -C 18 alkoxy, amino, mono-C 1 -C 18 alkylamino, di-C 1 -C 18 alkylamino, C 1 -C 18 alkylbenzylamino and C 1 -C 18 alkyl carboxamido.
  • aryl hydrocarbyl groups phenyl and naphthyl, as being exemplary of the group, with the understanding that the other enumerated aryl and substituted aryl R 1 groups can be utilized with substantially similar chemistry.
  • R 1 is phenyl, benzoyl chloride or benzoic anhydride can be used to form a preferred amide bond.
  • a benzenesulfonyl halide such as benzenesulfonyl chloride can similarly be used where Y is SO 2 .
  • Phenyl isocyanate is used where Y is HNC(O).
  • a phenyl chloroformate is used where Y is OC(O), whereas a phenyl chlorothioformate is used where Y is SC(O).
  • substituted phenyl R 1 groups include those in which the substituent can be substituted at any position of the ring, with the meta and para positions being preferred. Mono-substituted R 1 phenyl groups are preferred over di-substituted groups.
  • Contemplated halo substituents include fluoro, chloro, bromo and iodo groups, with p-fluorophenyl, m-chlorophenyl, m-iodophenyl, p-bromophenyl and
  • Exemplary C 1 -C 18 alkyl groups present as substituent groups on a phenyl of R 1 include straight and branched chain alkyl groups such as methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, t-butyl, pentyl, hexyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl and octadecyl.
  • C 1 -C 12 Alkyl groups are preferred, whereas C 1 -C 6 alkyl groups are particularly preferred, with methyl being most preferred.
  • Exemplary, preferred R 1 groups include o-, m- and p-tolyl (methylphenyl) and p-t-butylphenyl groups as well as 3,4-dimethylphenyl and 3,5-dimethylphenyl groups.
  • Exemplary C 1 -C 18 alkoxy groups are ethers containing a C 1 -C 18 alkyl group, or a particularly preferred C 1 -C 6 alkyl group. Methoxy is preferred here.
  • Exemplary, preferred R 1 groups include o, m- and p-anisyl (methoxyphenyl), as well as 3,4-dimethoxyphenyl and 3,5-dimethoxyphenyl.
  • a nitrophenyl R 1 group is readily prepared by acylation using 3- or 4-nitrobenzoyl chloride.
  • Acylation with 3,4- and 3,5-dinitrobenzoyl chloride provides the corresponding 3,4- and 3,5-dinitrophenyl R 1 groups.
  • Amide formation using 3- or 4-trifluoromethylbenzoyl chloride similarly provides 3- or 4-trifluoromethylphenyl R 1 groups.
  • a substituted phenyl R 1 group can also contain an amino, mono-C 1 -C 18 alkylamino, di-C 1 -C 18 alkylamino, benzylamino, C 1 -C 18 alkylbenzylamino or C 1 -C 18 alkyl carboxamido substituent, wherein C 1 -C 18 alkyl substituents are as discussed before.
  • Aminophenyl R 1 groups are most readily prepared from corresponding nitrophenyl R 1 groups discussed before by catalytic reduction of the nitro group after formation of the amide bond, as discussed before.
  • use of 3- or 4-nitrobenzoyl chloride to form the amide bond upon reduction with palladium on carbon forms the corresponding 3- or 4-aminophenyl R 1 group.
  • a similar use of 3,4- or 3,5-dinitrobenzoyl chloride provides the corresponding 3,4- or 3,5-diaminophenyl R 1 group after reduction.
  • di-C 1 -C 6 alkylaminobenzoic acids such as 4-diethylaminobenzoic acid and 3- and 4-dimethylaminobenzoic acids can be purchased commercially and used to form an appropriate benzoyl halide or anhydride for forming an R 1 -containing amide.
  • the remaining di-C 1 -C 18 alkylaminobenzoic acids and those compounds having two dialkylamino groups can be prepared using well known alkylation techniques from corresponding aminobenzoic acids or diaminobenzoic acids that are also commercially available.
  • a mono-C 1 -C 18 alkylaminophenyl R 1 group can be prepared from the corresponding mono-C 1 -C 18 alkylaminobenzoyl halide, whose remaining nitrogen valence is blocked by a readily removable blocking group such as t-Boc that can be removed with acid or a benzyl group that can be removed by hydrogenation, if desired, using palladium on carbon.
  • acylation can take place using N-benzyl-N-propylaminobenzoyl chloride, with the N-benzyl group being removed by catalytic hydrogenation to provide the mono-C 1 -C 18 alkylaminophenyl R 1 group.
  • the benzyl group need not be removed, thereby providing a C 1 -C 18 alkylbenzylamino group.
  • Each of the above-discussed phenyl or substituted phenyl substituents can be prepared by a well known amide-forming reaction.
  • An exemplary reaction reacts an appropriate benzoyl halide or anhydride such as E-fluorobenzoyl chloride or benzoic anhydride with the unprotected amine group of an otherwise protected saccharide as is illustrated in detail hereinafter.
  • Both 1- and 2-naphthyl R 1 groups are contemplated, with 2-naphthyl being particularly preferred.
  • These compounds can also be prepared using standard amide- forming technology as above, such as by reacting 2-naphthoyl chloride with an amine of an appropriate saccharide as discussed above.
  • substituents are present on the oxa-, thia- , aza- and diazahydrocarbyl aryl groups.
  • Y is SO 2
  • a corresponding sulfonyl halide is used.
  • benzenesulfonyl chloride toluenesulfonyl chloride
  • the isocyanate corresponding to a before-described carboxylic acid is a convenient reactant.
  • Such derivatives can be readily prepared from the acid halide by reaction with azide, to form the acyl azide, which undergoes the Curtius rearrangement to form the isocyanate upon heating.
  • Y is OC(O) or SC(O)
  • a hydroxyl or mercapto substituted aryl R 1 group is reacted with phosgene to form the chloroformate or chlorothioformate that can be reacted with the saccharide amine to form the urethane or thiourethane linkage to an
  • a phenyl C 1 -C 3 alkylene R 1 group is a C 1 -C 3 alkylene group that is itself substituted with a phenyl group, preferably at the terminal hydrocarbyl group carbon.
  • This R 1 C(O) group thus contains a phenyl ring linked to a chain of 2-4 carbon atoms.
  • These compounds can be prepared by reaction of an appropriate acid halide or anhydride with a saccharidal amine as above. Catalytic reduction using hydrogen and a palladium on carbon catalyst can be used to form saturated alkylene groups from the unsaturated hydrocarbyl chains; saturated hydrocarbyl chains being preferred.
  • R 2 group forms a ⁇ -glycoside with the saccharide ring system.
  • That glycoside bond can be formed from a simple C 1 -C 18 hydrocarbyl alcohol, from an ⁇ -hydroxycarboxylic acid ester, from an ⁇ -hydroxylated silylated alkyl group, or from a mono- or a disaccharide, or OR 2 together form a C 1 -C 18 straight chain, branched chain or cyclic hydrocarbyl carbamate.
  • a C 1 -C 6 hydrocarbyl group such as ethyl, a benzyl group or a monosaccharide such as 3-galactosyl is particularly preferred.
  • R 2 can also be hydrogen.
  • R 2 groups formed from simple precursor alcohol groups include C 1 -C 18 straight chain, branched chain or cyclic hydrocarbyl groups.
  • Illustrative of such groups are the before-described C 1 -C 6 alkyl groups, which are preferred, as well as their unsaturated counterparts, such as allyl, 3-butenyl, 2-but-3-enyl, and but-3-ynyl, as well as longer hydrocarbyl groups such as benzyl, 4-methylcyclohexyl, decahydronaphthyl, nonyl, decyl (capryl), dodecyl (lauryl), dodec-7-enyl, myristyl, palmityl, stearyl, oleyl, linoleyl, linolenyl and ricinoleyl.
  • a C 1 -C 18 hydrocarbyl carbamate is prepared by reaction of an isocyanate corresponding to a before discussed C 1 -C 18 hydrocarbyl group with the hydroxyl group of the reducing end sugar.
  • the 1 -hydroxyl group of a terminal glucosyl unit can be reacted with ethylisocyanate to form the corresponding ethyl carbamate
  • the carbonyl group of the carbamate is not included in the number of hydrocarbyl carbon atoms.
  • a C 1 -C 6 alkyl C 1 -C 5 -alkylene ⁇ -carboxylate R 2 group is a C 1 -C 6 alkyl ester of a C 2 -C 6 ⁇ -carboxylic acid.
  • esters are prepared from precursor
  • ⁇ -hydroxycarboxylic acid esters whose hydroxyl groups are used to form the glycosidic bond.
  • Exemplary ⁇ -hydroxycarboxylate esters include methyl 2-hydroxyacetate, ethyl 3-hydroxypropionate, t-butyl 4-hydroxybutyrate, hexyl 5-hydroxypentanoate and methyl 6-hydroxyhexanoate.
  • the hydroxyl and carboxyl groups are at the termini of the chain and are separated by 1-5 methylene groups.
  • Methyl 6-hydroxyhexanoate acid is preferred.
  • An ⁇ -tri(C 1 -C 4 alkyl/phenyl)silyl C 2 -C 4 alkyl R 2 group is formed from a corresponding precursor alcohol whose substituted silyl group is at the terminus
  • substituted silyl groups can include many combinations of C 1 -C 4 alkyl and phenyl groups such as tri-C 1 -C 6 alkyl, di-C 1 -C 4 alkylphenyl, C 1 -C 4 alkyldiphenyl and triphenyl.
  • Exemplary substituted silyl groups include trimethylsilyl, triphenylsilyl,
  • Exemplary mono- and disaccharides include 3- and 4-glucosyl (3/4Glc), 3-and 4-galactosyl (3/4Gal), a 3-galactosyl group being particularly preferred, 3- and 4-N-acetylglucosyl (3/4GlcNAc), 2, 3-, 4- and 6-mannosyl (2/3/4/6Man), and 3- and 6-N-acetylgalactosyl (3/6 GalNAc) and Gal ⁇ 1 ⁇ 4GlcNAc.
  • a monosaccharide can itself form a glycoside linkage with a group, R 6 , that includes all but a saccharide of an R 2 group.
  • R 6 is R 2 other than mono- or disaccharide.
  • a structural formula for a particularly preferred compound of Formula A having a reducing terminal 3Gal ⁇ OR 6 group is shown below in structural Formula B wherein X, Y, Z and R 1-4 , R 6 and R 7 are as defined before.
  • a particularly preferred compound of Formula B is an inhibitor of cellular adhesion having a structure of Formula C, below, wherein X, Y, R 1 , R 6 and R 7 are as before disclosed.
  • the ⁇ -glycosyl bond formed with an R 2 or R 6 group can be prepared by well known organic chemical reactions with both the saccharides and other R 2 (R 6 ) group precursors, as by reaction of a 1-halo saccharide with a hydroxyl of a desired R 2 (R 6 ) group precursor alcohol in the presence of silver carbonate (Ag 2 CO 3 ) or silver triflate, as well as by enzymatic means as with a glycosyl transferase for the saccharides.
  • a contemplated R 3 group can be hydrogen or C 1 -C 6 acyl, which is the acid portion of a C 1 -C 6 acyl carboxylic acid ester.
  • a C 1 -C 6 acyl group is preferred for a compound of Formula II.
  • Exemplary C 1 -C 6 acyl groups include formyl, acetyl, propionyl, butanoyl, isobutanoyl, pentanoyl and hexanoyl.
  • An acetyl group is preferred.
  • Acylation of saccharide hydroxyl groups is well known and can be carried out using an appropriate acid halide or anhydride.
  • a contemplated R 4 group of Formula A can be hydrogen, a C 1 -C 6 alkyl, as was discussed before for such alkyl groups, or a benzyl group.
  • An R 4 group along with its bonded oxygen atom forms the alcohol portion of an ester.
  • a methyl group is preferred.
  • the R 4 ester can be formed by standard means prior to the addition of the sialic acid group, after formation of the sialylated saccharide using a reagent such as diazomethane, or by reaction of a lactone with an appropriate alcohol as discussed in regard to Scheme 2, hereinafter.
  • the R 4 group of a compound of Formula III can be either a proton, C 1 -C 6 alkyl or benzyl groups with C 1 -C 6 alkyl being preferred.
  • R 4 is present as a proton, it is to be understood that that proton can be replaced by a pharmaceutically acceptable cation (M) such as ammonium, sodium, potassium, calcium, magnesium and the like.
  • M pharmaceutically acceptable cation
  • the R 4 proton or other cation is typically not shown in the structures herein such as Formulas I and C because the sialyl carboxylic acid is usually ionized at physiological pH values of about 7.2-7.4 at which an inhibitor of Formulas I or C is utilized. Thus, the sialyl carboxyl group is often shown herein as a carboxylate.
  • R 5 group is a hydrogen, a benzyl, methoxybenzyl (3- or 4- methoxybenzyl being preferred), a dimethoxybenzyl such as 3,4- or 3,5- dimethoxybenzyl, or a C 1 -C 6 acyl group as discussed previously.
  • a benzyl group is usually used where the fucosyl group is added by organic chemical synthesis.
  • R 3 , R 4 and R 5 groups other than hydrogen are protecting groups used during synthesis of intermediates such as a compound of Formulas B, II and III, above.
  • a compound of Formula H becomes a compound of Formula I
  • a compound of Formula B becomes a compound of Formula C
  • Z fuco
  • a compound of Formula A becomes a compound of Formula I.
  • An X substituent group can be a C 1 -C 6 acyloxy group; i.e., a C 1 -C 6 acyl ester of a precursor hydroxyl group at that position, a C 2 -C 6 hydroxylacyloxy group, a hydroxyl group, a halo group, as discussed previously, or an azido group.
  • Exemplary C 1 -C 6 acyl groups have already been discussed, and a C 1 -C 6 acyloxy group is a C 1 -C 6 acyl group that further includes an additional oxygen atom bonded to the carbonyl carbon atom of an acyl group.
  • a C 2 -C 6 hydroxylacyloxy group is an above-discussed C 1 -C 6 acyloxy group that further includes a substituent hydroxyl group.
  • Exemplary C 2 -C 6 hydroxylacyloxy groups include hydroxyacetate, lactate, 3-hydroxybutyrate,
  • An X substituent is usually other than C 1 -C 6 acyloxy or C 2 -C 6 hydroxylacyloxy unless both sialylation and fucosylation are carried out enzymatically, as is discussed hereinafter.
  • R 7 group is methyl or hydroxy methyl, so that along with the depicted carbonyl group [C(O)] R 7 forms an N-acetyl or N-hydroxyacetyl group.
  • Sialic acid derivatives containing either R 7 group can be used in an enzymatic sialylation as described herein.
  • a before-described SLe x analogue compound can be prepared in numerous ways. Thus, completely enzymatic syntheses can be carried out, syntheses using only the techniques of organic chemistry can be used, and mixtures of both organic and enzymatic syntheses can be utilized, as is exemplified here.
  • One way to distinguish between organic and enzymatic syntheses is by the presence of one or more enzymes in a water-based reaction medium (enzymatic synthesis), versus the absence of any enzymes coupled with a reaction medium that is substantially free of water and utilizes an organic solvent such as acetonitrile, methanol, ethanol, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), benzene, acetone, dichloromethane, tetrahydrofuran (THF) and the like (organic synthesis).
  • an organic solvent such as acetonitrile, methanol, ethanol, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), benzene, acetone, dichloromethane, tetrahydrofuran (THF) and the like (organic synthesis).
  • the saccharides comprising lactosamine, galactose and glucosamine, must be joined together at some point in the syntheses.
  • the Gal ⁇ 1 ⁇ 4GlcN bond of lactosamine is also one of the more difficult bonds to form in the synthesis of a contemplated compound.
  • Lactosamine is a compound reported in the literature, but is not readily available. Nevertheless, lactosamine or a derivative of lactosamine provides a good starting material for synthesis of a contemplated compound.
  • lactosamine is not readily available
  • lact ⁇ lose a ketose that possesses no amine group but contains a Gal ⁇ 1 ⁇ 4Fru bond that is related to lactose and lactosamine
  • Lactulose with its Gal ⁇ 1 ⁇ 4 bond already formed, provides a starting material for one contemplated synthesis of lactosamine.
  • a synthesis of lactosamine (Compound 3) as an acid addition salt is illustrated generally and specifically below in Schemes 1 and 1A, respectively, as are the syntheses of peracetyl N-phthalimidolactosamine (Compound 5) and peracetyl
  • N-phthalimidolactosamine ⁇ chloride (Compound 6). Numbered compounds in both schemes are the same compounds.
  • Reaction of Compound 1 in methanol with about a stoichiometric amount of an organic carboxylic acid having a pK a value of about 2.5 to about 5.0 (glacial acetic acid) provided N-benzyl lactosammonium acetate (Compound 2) in 50-55 percent yield.
  • Lactosammonium acetate (Compound 3) was prepared by hydrogenolysis of the above methanolic solution using palladium on carbon (Pd/C).
  • reductively removable blocked amines can be used in place of benzylamine.
  • mono- and dimethoxybenzylamines can be viewed as reductively removable blocked ammonia derivatives in that after reaction with the saccharide, the mono- and dimethoxybenzyl groups can also be removed by
  • Allylamine can similarly be used, with the allyl blocking group being removed by reaction with polymethylhydrosiloxane (PMSH) and palladium-tetrakistriphenylphosphine [Pd(PPh 3 ) 4 ] in THF as solvent.
  • PMSH polymethylhydrosiloxane
  • Pd(PPh 3 ) 4 palladium-tetrakistriphenylphosphine
  • the blocked ammonia derivative (or primary amine) is present in a 2- to about 10-fold molar excess over the moles of lactuiose utilized.
  • the primary amine is preferably present in about a 4- to about 8-fold molar excess.
  • N-glycoside to form i.e., for the primary amine to replace the lactuiose 2-hydroxyl group.
  • Temperatures from ambient room temperature (about 20°C) to about 50°C are preferred.
  • the maintenance time is a function of several variables such as the molar excess of primary amine, maintenance temperature, and the amount of lactuiose
  • N-glycoside desired can range from about 8 hours, where little of the product is desired, to as much as two weeks, using low temperatures and amounts of primary amine.
  • primary amine here, benzylamine
  • the reaction was complete after a maintenance time of seven days at room temperature, but less than 50 percent complete over the same time when a 2-fold excess of benzylamine was used under the same conditions.
  • the maintenance temperature was raised to 50°C, the reaction using a 4-fold excess of amine was complete after two days (48 hours), whereas a 70°C reaction temperature caused decomposition.
  • Lactuiose is insoluble in alcohol solvents, including methanol. Lactuiose can be dissolved in hot DMF and remain in solution after cooling. Both methanol and DMF can be used as cosolvents with the primary amine when an above-discussed catalyst is also present. For example, when methanol was used as a cosolvent, no reaction was had at either room temperature or 50°C. However, when a zinc chloride catalyst was used with a 4-fold excess of benzylamine and methanol as cosolvent, the reaction was complete after 48 hours at room temperature.
  • the lactuiose N-glycoside prepared as discussed above is hygroscopic, and is therefore used quickly after its preparation. That N-glycoside is reacted with about 0.1 equivalents up to an equivalent amount (for best yield) of a carboxylic acid having a pK a value of about 2.5 to about 5.0 in a C 1 -C 3 alcohol solvent at a temperature of about 10°C to about 30°C to rearrange the lactuiose N-glycoside into a lactosammonium salt whose amine group is blocked with an above reductively removable blocking group; i.e., an amine-blocked lactosammonium salt having a reductively removable blocking group bonded to the amine nitrogen atom.
  • Exemplary C 1 -C 3 alcohols include methanol, which is preferred, ethanol, propanol and iso-propanol.
  • concentration of lactuiose N-glycoside can range from about 0.1 M to substantial saturation. Typically utilized concentrations are about 0.5 to about 1.5 M in the solvent.
  • the reductively removable blocking group is then removed.
  • Hydrogenolysis using a palladium catalyst is a preferred process for that removal, particularly where benzylamine or a methoxybenzylamine is used.
  • PMHS and Pd(PPh 3 ) 4 are used where allylamine is the primary amine.
  • the above reduction can take place in any appropriate solvent for the lactosammonium derivative.
  • hydrogenolysis can be carried out in acidic water or C 1 -C 3 alcohol as above.
  • PMHS and Pd(PPh 3 ) 4 are typically utilized in THF or a similar solvent.
  • a thus produced lactosammonium salt is generally recovered after preparation, although, depending upon the solvent used and the use to be put to the compound, recovery is not necessary. Where it is desired to recover the
  • lactosammonium salt whose anion is the anion form of the acid used in the reduction, can be obtained by well known methods such as chromatography or precipitation.
  • Free lactosamine can be prepared from the salt by ion exchange chromatography or by neutralization, followed by extraction of the free base into an appropriate organic solvent.
  • lactuiose is reacted in a stainless steel autoclave with an equimolar amount of ammonium acetate and liquid ammonia as solvent, the liquid ammonia being added to the autoclave cooled to -78°C.
  • the resulting reaction mixture is warmed to a temperature from zero degrees C to about 80°C, and maintained for a period of about five hours to about five days, depending upon the temperature used and desired conversion.
  • This reaction forms lactuiose aminoglycoside.
  • the resulting ammonia- free material is treated with a carboxylic acid as before to form the lactosammonium salt, e.g. Compound 3.
  • the lactosammonium salt is also treated as discussed before to form Compound 5.
  • the ⁇ -anomer of Compound 5 was recovered in 3.8 percent overall yield in the first crystallization, where a reaction temperature of 35°C and reaction time of 24 hours was utilized in the first reaction step.
  • the amine of Compound 5 in Scheme 1 is shown bonded to R B and R B groups that together with the depicted nitrogen atom form a C 4 -C 8 cyclic imide such as an exemplary phthalimide (Phth) in Compound 5.
  • a C 4 -C 8 cyclic imide such as an exemplary phthalimide (Phth) in Compound 5.
  • succinic anhydride, maleic anhydride, mono- and dimethylsuccinic anhydrides and citraconic anhydride can also be used to form similar imides, so that R B -and R B together with the nitrogen atom form a corresponding imide.
  • a cyclic imide formed by the -NR B R B group provides an amine protecting group that is stable under conditions in which O-acyl groups such as acetate are removed, but can be readily removed with hydrazine.
  • an anhydride need not be used, but can be replaced by a C 1 -C 6 alkyl half ester halide such as
  • Compound 5 is shown as the ⁇ -anomer.
  • the ⁇ -acetate is also formed and the yield of the desired ⁇ -acetate can be almost doubled by concentrating the mother liquor from which Compound 5 was obtained to a foam followed by redissolution in DMF and then reaction with hydrazinium acetate, which cleaved the acetate group and caused formation of the ⁇ -OH anomer.
  • hydrazinium acetate which cleaved the acetate group and caused formation of the ⁇ -OH anomer.
  • an additional 8.3 percent overall percent yield of Compound 5 was obtained.
  • the final yield of Compound 5 of 18.7 percent was obtained, based on starting materials.
  • Scheme 2 hereinafter, illustrates the transformation of Compound 6, peracetyl N-phthalimidolactosamine ⁇ -chloride, into the fully protected sialylated tetrasaccharide, Compound 13.
  • Compound 6 was reacted at ambient temperature for two hours in step a with Compound 9, whose synthesis is discussed in the examples, in the presence of molecular sieves, collidine and silver trifluoromethanesulfonate (triflate) using dichloromethane as solvent to prepare the corresponding trisaccharide.
  • diallylpyrocarbonate in step d provided Compound 10, where AL is allylcarbamoyl.
  • R 2 is not a glycoside as described in the syntheses of Scheme 2, and is rather a preferred C 1 -C 18 hydrocarbyl group such as benzyl, the glycosylation steps a and b are omitted, providing a tetrasaccharide of Formulas A, I or II, where R 2 is other than mono- or disaccharide.
  • Compound 10 was then sialylated enzymatically in step e in an aqueous buffer using ⁇ -(2,3)-sialyltransferase (EC 2.4.99.6) and a number of other enzymes. The reaction was followed by TLC for 10-12 days at ambient temperature, at which time more than 95 percent of Compound 10 had been consumed, and Compound 11 was prepared.
  • Compound 11 was recovered as a thick syrup that was coevaporated twice with pyridine and then kept under vacuum for 20 hours. The thus dewatered material was redissolved in pyridine to which a catalytic amount of 4-dimethylaminopyridine (DMAP) was added as was acetic anhydride. Two more additions of acetic anhydride over the ensuing 44 hours completed the acetylation reaction and formation of a lactone with the sialyl carboxyl and a saccharide hydroxyl in step f. Methanol was thereafter added to the recovered material to form the sialyl methyl ester and thereafter, another addition of acetic anhydride was made to acetylate the freed hydroxyl to form completely protected Compound 12 in step g.
  • DMAP 4-dimethylaminopyridine
  • Compounds 11 and 12 are compounds of structural Formulas A and III.
  • Z is C 1 -C 6 acyl (acetyl)
  • X is C 1 -C 6 acyloxy (acetoxy)
  • R 2 is 3Gal ⁇ O-ethyl
  • R 3 is acetyl
  • R 4 is methyl
  • R 1 is allyloxy.
  • sialyl unit X substituents that are C 1 -C 6 acyloxy or C 1 -C 6 hydroxylacyloxy be present in an inhibitor of structural Formulas I or C
  • Compound 10 or a disaccharide without the 3Gal ⁇ OR 2 group
  • the allyloxy carbamoyl group (AL) of Compound 10 be removed as in step h, and replaced by one of the phenyl ring-containing R 1 acyl groups as in step c of Scheme 3.
  • the molecule is then deprotected and enzymatically sialylated and
  • Compound 14 was then selectively benzoylated in step b in 83 percent yield by reaction with benzoyl chloride in dichloromethane with solid sodium bicarbonate at room temperature for 24 hours to form Compound 15.
  • the alterative R 1 groups of a compound of structural Formulas A, I, II and III are added at this step or at an analogous step where R 2 is not a saccharide unit.
  • step c of Scheme 3 An organic chemical fucosylation was carried out in step c of Scheme 3 by mixing Compound 15 with tri-O-benzyl fucosyl fluoride, molecular sieves and
  • Compound 16 is thus a compound of structural Formulas A and B, where Z is a blocked fucosyl group, as well as a compound of Formula II.
  • Use of alternative R 5 groups provide the remaining compounds of those structural formulas when combined with the before-discussed X and R 1-4 groups.
  • the O-benzyl blocking groups, R 5 , of the fucosyl saccharide unit were removed in step d by hydrogenation using palladium hydroxide on carbon [Pd(OH) 2 /C] in methanol as solvent. Reaction for one hour at room temperature provided complete removal of the O-benzyl groups. Filtration and concentration of the debenzylated compound provided an oil that was redissolved in methanol: water (4:1) to which was added sodium methoxide powder in step e. After 16 hours of reaction at room
  • R 5 is a C 1 -C 6 acyl group
  • the hydrogenation step is not used and the R 5 C 1 -C 6 acyl group is removed along with the R 3 and R 4 groups.
  • Use of an R 5 C 1 -C 6 acyl group and the avoidance of a hydrogenation step also provides a route for synthesis of nitro group-containing R 1 groups.
  • the R 2 group is a mono- or disaccharide
  • an appropriately blocked mono- or disaccharide is used such as Compound 9 of Scheme 2.
  • lactose a lactose C 1 -C 18 glycoside or melibiose
  • a lactose C 1 -C 18 glycoside or melibiose can be made into protected (blocked) benzylidine derivatives similar to that of Compound 9 and then used in the coupling step a of Scheme 2, and the resulting product used in subsequent steps of Schemes 2 and 3.
  • lactosamine and its derivatives can be prepared by other methods well known to skilled workers. It is to be further understood that the trisaccharide Compound 10 can be prepared enzymatically by reaction of ethyl
  • Compound 11 can be fucosylated enzymatically using a fucosyl transferase (FT), such as fucosyl transferase V, as well as the nucleotide sugar donor GDP-fucose, and other enzymes useful in the regeneration of GDP-fucose, using known procedures.
  • FT fucosyl transferase
  • Step a of Scheme 4 is substantially the same glycosylation step shown as step c of Scheme 3, with Compound 40 being formed in 73 percent yield, plus recovery of 17 percent starting Compound 39.
  • the fucosylated free amine, Compound 41 was thereafter formed in 96 percent yield in step c by reaction with ten percent Pd-C in ammonium formate in ethanol at reflux.
  • the free amine of Compound 41 was thereafter reacted in step d with an acyl (YR 1 ) chloride in dichloromethane in the presence of sodium bicarbonate to provide the corresponding hydroxy-blocked N-acylated compound, here, the 3,5-dichlorbenzamide derivative, Compound 42, in high yield.
  • the hydroxyl groups were de-blocked by reaction in 28 percent sodium methoxide-methanol in substantially quantitative yield.
  • a pharmaceutical composition containing a contemplated SLe x analogue compound dissolved or dispersed in a pharmaceutically acceptable carrier or diluent is also contemplated.
  • Such a composition contains a cell adhesion-inhibiting amount of a before-discussed, contemplated SLe x analogue compound.
  • compositions which are SLe 1 analogue compounds, liposomal formulations of a lipid-SLe x conjugate (e.g., liposomal formulations of compounds of formula IV in which either R 1 or R 2 is a lipid or a linking group with an attached lipid), and liposomal formulations of a lipid-SLe x conjugate in which the liposome portion comprises additional therapeutic agent(s).
  • the SLe x portion of the liposome formulation can serve as a pharmaceutical agent in combination with an additional encapsulated drug, or it can serve as a targeting agent for the liposome formulation, or it can provide dual advantage of both a pharmaceutical agent and a targeting agent.
  • any reference to the SLe x portion as a targeting agent is not meant to be limiting on its actual function in vivo.
  • Each of the pharmaceutical compositions has utility as a pharmaceutical preparation which will be understood by those of skill in the art, in the context of the disclosure below.
  • a cellular adhesion-inhibiting amount can vary widely. That amount is, however, sufficient to inhibit binding of cells that express sialyl Le x on their cell surfaces to selectin, particularly E-selectin (ELAM-1) preferably by about one-half or more.
  • An exemplary cellular adhesion-inhibiting amount is about 5 to about 60 mg/kg.
  • a contemplated pharmaceutical composition can be used to block or inhibit cellular adhesion associated with a number of disorders.
  • a number of inflammatory disorders are associated with selectins expressed on vascular endothelial cells and platelets.
  • the term "inflammation" is used here to refer to reactions of both the specific and non-specific defense systems.
  • a specific defense system reaction is a specific immune system reaction to an antigen.
  • Exemplary of specific defense system reactions include antibody response to antigens, such as viruses, and delayed-type hypersensitivity.
  • a non-specific defense system reaction is an inflammatory response mediated by leukocytes generally incapable of immunological memory. Such cells include macrophages, eosinophils and neutrophils.
  • non-specific reactions include the immediate swelling after a bee sting, and the collection of peripheral mononuclear (PMN) leukocytes at sites of bacterial infection (e.g., pulmonary infiltrates in bacterial pneumonia and pus formation in abscesses).
  • PMN peripheral mononuclear
  • treatable disorders include, e.g., rheumatoid arthritis, post-ischemic leukocyte-mediated tissue damage (reperfusion injury), frost-bite injury or shock, acute leukocyte-mediated lung injury (e.g., adult respiratory distress syndrome), asthma, traumatic shock, septic shock, nephritis, and acute and chronic inflammation, including atopic dermatitis, psoriasis, and inflammatory bowel disease.
  • Various platelet-mediated pathologies such as atherosclerosis and clotting can also be treated.
  • tumor metastasis can be inhibited or prevented by inhibiting the adhesion of circulating cancer cells. Examples include carcinoma of the colon and melanoma.
  • reperfusion injury is particularly amenable to treatment by a contemplated pharmaceutical composition.
  • a composition that inhibits a P-selectin-ligand interaction can be particularly useful for treating or preventing reperfusion injury.
  • a contemplated pharmaceutical composition can be used
  • inhibitors of the P-selectin-ligand interaction can be especially useful in minimizing tissue damage that often accompanies thrombotic disorders. For instance, such inhibitors can be of therapeutic value in patients who have recently experienced stroke, myocardial infarctions, deep vein thrombosis, pulmonary embolism, etc.
  • the compounds are especially useful in pre-thrombolytic therapy.
  • a contemplated composition finds particular use in treating the secondary effects of septic shock or disseminated intravascular coagulation (DIC).
  • DIC disseminated intravascular coagulation
  • Leukocyte emigration into tissues during septic shock or DIC often results in pathological tissue destruction. Furthermore, these patients can have widespread microcirculatory thrombi and diffuse inflammation.
  • a therapeutic composition provided herein inhibits leukocyte emigration at these sites and mitigates tissue damage.
  • An inhibitor of a selectin-cellular SLe x ligand interaction is also useful in treating traumatic shock and acute tissue injury associated therewith. Because the selectins play a role in recruitment of leukocytes to the sites of injury, particularly
  • E-selectin in cases of acute injury and inflammation, inhibitors thereof can be administered locally or systemically to control tissue damage associated with such injuries. Moreover, because of the specificity of such inhibitors for sites of
  • these compositions can be more effective and less likely to cause complications when compared to traditional anti- inflammatory agents.
  • the present invention also provides a pharmaceutical composition that can be used in treating the aforementioned conditions.
  • composition is comprised of a before-described SLe x analogue compound that inhibits the interaction between a cellular SLe x ligand and a selectin receptor, which compound is dissolved or dispersed in a pharmaceutically acceptable diluent.
  • a contemplated pharmaceutical composition is suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see, Langer, Science, 249: 1527-1533 (1990).
  • a contemplated pharmaceutical composition can further include other compounds known to interfere with the function of other cellular adhesion molecules.
  • members of the integrin family of adhesion molecules are thought to play a role in the extravasation of leukocytes at points of infection.
  • intercellular adhesion receptors including selectin receptors, and their role immune function, see Springer, Nature, 346:425-434 (1990).
  • successful treatment using a contemplated pharmaceutical composition can also be determined by the state of development of the condition to be treated. Because different adhesion molecules can be up or down regulated in response to a variety of factors during the course of the disease or condition, one of skill will recognize that different
  • compositions can be required for treatment of different inflammatory states.
  • a before-described SLe x analogue compound of the pharmaceutical composition can be used to target conventional anti-inflammatory drugs or other agents to specific sites of tissue injury.
  • a drug to a selectin receptor on, e.g., a vascular endothelial cell such drugs can achieve higher concentrations at sites of injury.
  • Side effects from the conventional anti-inflammatory chemotherapeutic agents can be substantially alleviated by the lower dosages, the localization of the agent at the injury sites and/or the encapsulation of the agent prior to delivery.
  • the targeting component i.e., the SLe x analogue compound that binds to a selectin
  • the coupling which can be performed by means, generally known in the art, should not substantially inhibit the ability of the ligand to bind the receptor nor should it
  • chemotherapeutics can be coupled for targeting.
  • anti-inflammatory agents that can be coupled include immunomodulators, platelet activating factor (PAF) antagonists, cyclooxygenase inhibitors, lipoxygenase inhibitors, and leukotriene antagonists.
  • Some preferred moieties include cyclosporin A, indomethacin, naproxen, FK-506, mycophenolic acid, etc.
  • anti-oxidants e.g., superoxide dismutase, are useful in treating reperfusion injury when targeted by a contemplated saccharide compound.
  • anticancer agents can be targeted by coupling the SLe x analogue compound to the chemotherapeutic agent.
  • agents that can be coupled include daunomycin, doxorubicin, vinblastine, bleomycin, etc.
  • a C 1 -C 6 alkyl C 1 -C 5 alkylene ⁇ -carboxylate R 1 group can be used for coupling.
  • the selectin receptor targeting can also be accomplished via amphipaths, or dual character molecules (polar: nonpolar) that exist as aggregates in aqueous solution.
  • Amphipaths include nonpolar lipids, polar lipids, mono- and diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids and salts. These molecules can exist as emulsions and foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions and lamellar layers. These are generically referred to herein as liposomes. In these preparations the drug to be delivered is incorporated as part of a liposome in conjunction with a SLe x analogue compound that binds to the selectin receptor.
  • a contemplated SLe x analogue compound whose R 2 group is a C 12 -C 18 hydrocarbyl group is particularly useful in such liposome preparations.
  • Still other contemplated SLe x analogue compounds are those in which either R 1 or R 2 is a lipid or a linking group with an attached lipid. Methods of attaching lipids to oligosaccharides as well as suitable lipids for use in such conjugates are described in PCT/US94/03103 (WO 94/21235), incorporated herein by reference.
  • liposomes filled with a desired chemotherapeutic agent can be directed to a site of tissue injury by the selectin-SLe x analogue compound interaction. When the liposomes are brought into proximity of the affected cells, they deliver the selected therapeutic compositions.
  • the liposomes of the present invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • the selection of lipids is generally guided by consideration of, e.g. , liposome size and stability of the liposomes in the bloodstream.
  • the major lipid component in the liposomes is
  • phosphatidylcholine phosphatidylcholines having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. In general, less saturated phosphatidylcholines are more easily sized, particularly when the liposomes must be sized below about 0.3 microns, for purposes of filter sterilization. Methods used in sizing and filter-sterilizing liposomes are discussed below.
  • the acyl chain composition of phospholipid can also affect the stability of liposomes in the blood.
  • One preferred phosphatidylcholine is partially hydrogenated egg phosphatidylcholine.
  • Targeting mechanisms generally require that the targeting agents be positioned on the surface of the liposome in such a manner that the target agents are available for interaction with the selectin receptor.
  • the liposome is typically fashioned in such a way that a connector portion is first
  • the connector portion has a lipophilic portion that is firmly embedded and anchored in the membrane. It also has a hydrophilic portion that is chemically available on the aqueous surface of the liposome. The hydrophilic portion is selected so that it is chemically suitable to form a stable chemical bond with the targeting agent (e.g., SLe x analog) which is added later. Therefore, the connector molecule has both a lipophilic anchor and a hydrophilic reactive group suitable for reacting with the SLe x analog and holding the SLe x analog in its correct position, extended out from the liposome' s surface.
  • the targeting agent e.g., SLe x analog
  • the targeting agent (SLe x analog) is conjugated to a lipid, typically a phospholipid, and then brought into contact with a liposome.
  • a lipid typically a phospholipid
  • Examples of SLe 1 analogs which are covalently attached to a phospholipid are provided in Examples 30 and 31, below.
  • a phosphatidyl thioethanol was coupled (in Example 30) to a pentasaccharide bearing a reactive ⁇ -bromoacetamide (see Figure 6).
  • compounds 62 see Figure 1), 73 (see Figure 2), 78 (see Figure 3), 85 (see Figure 4), and 100 (see Figure 5) have also been coupled to a phospholipid (see Example 31).
  • other phospholipids bearing suitable nucleophilic functionality could also be coupled with the saccharides bearing a reactive ⁇ -bromoacetamide groups.
  • other methods of coupling a phospholipid to a SLe x analog could be employed.
  • diacylphosphatidylethanolamine can be coupled to a SLe x analog bearing a suitable carboxylic acid or ester in an amide-forming reaction.
  • other suitable bond-forming reactions could be employed to link a phospholipid to a SLe x analog via a new ester, ether, disulfide, thioether, or hydrazone.
  • the phospholipids or other lipid species which are conjugated to a SLe x analog can have fatty acid side chains of from about 8 to about 24 carbons in length which are saturated or unsaturated.
  • the position of the SLe x analogue on the surface of the liposome can be adjusted and controlled by the length of the spacer between the analogue and the phospholipid.
  • compound 114 has a propylene group which connects the pentasaccharide portion and the ⁇ -bromoacetamide portion.
  • Alternative groups such as longer alkylene chains (e.g., — (CH 2 ) 5 —,—(CH 2 ) 10 —,—(CH 2 ) 14 — and— (CH 2 ) 20 —) could also be used.
  • the SLe x analogue can be positioned on the surface of the liposome and outside the area occupied by any circulation enhancing agents (e.g., polyethylene glycols or polyethylene glycol-lipid conjugates).
  • Liposome charge is an important determinant in liposome clearance from the blood, with negatively charged liposomes being taken up more rapidly by the reticuloendothelial system (see Juliano, Biochem. Biophys. Res. Commun., 63:651 (1975)) and thus having shorter half-lives in the bloodstream. Liposomes with prolonged circulation half-lives are typically desirable for therapeutic and diagnostic uses.
  • the liposomes are prepared with about 5-15 mole percent negatively charged phospholipids, such as phosphatidylglycerol, phosphatidylserine or phosphatidylinositol.
  • negatively charged phospholipids such as phosphatidylglycerol, phosphatidylserine or phosphatidylinositol.
  • the added negatively charged phospholipids such as phosphatidylglycerol, phosphatidylserine or phosphatidylinositol.
  • phosphatidylglycerol serve to prevent spontaneous liposome aggregating, and thus minimize the risk of undersized liposomal aggregate formation.
  • Membrane-rigidifying agents such as sphingomyelin or a saturated neutral phospholipid, at a concentration of at least about 50 mole percent, and 5-15 mole percent of monosialylganglioside, can provide increased circulation of the liposome preparation in the bloodstream, as generally described in U.S. Pat. No. 4,837,028, incorporated herein by reference.
  • the liposome suspension can include lipid-protective agents that protect lipids and drug components against free-radical and lipid-peroxidative damages on storage.
  • Lipophilic free-radical quenchers such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxianine, are preferred.
  • One method produces multilamellar vesicles of heterogeneous sizes.
  • the vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or an inert gas to form a thin lipid film.
  • the film can be redissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogeneous lipid mixture that is in a more easily hydrated powder-like form.
  • This film is covered with an aqueous solution of the targeted drug and the targeting component and allowed to hydrate, typically over a 15-60 minute period with agitation.
  • the size distribution of the resulting multilamellar vesicles can be shifted toward smaller sizes by hydrating the lipids under more vigorous agitation conditions or by adding solubilizing detergents such as deoxycholate.
  • the hydration medium contains the targeted drug at a concentration that is desired in the interior volume of the liposomes in the final liposome suspension.
  • the drug solution contains between 10-100 mg/mL in a buffered saline.
  • concentration of the targeting SLe x analogue compound which binds a selectin is generally between about 0.05 - 20 mg/mL.
  • the liposomes can be sized to achieve a desired size range and relatively narrow distribution of liposome sizes.
  • One preferred size range is about 0.1-0.2 microns, which allows the liposome suspension to be sterilized by filtration through a conventional filter, typically a 0.22 micron filter.
  • the filter sterilization method can be carried out on a high through-put basis if the liposomes have been sized down to about 0.1-0.2 microns.
  • Extrusion of liposome through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing liposome sizes to a relatively well-defined size distribution.
  • the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved.
  • the liposomes can be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.
  • the initial sized liposome suspension can contain up to 50 percent or more drug or targeting agent (lipid-SLe x conjugate) in free form (e.g., non-encapsulated or unincorporated) . Therefore, to maximize the advantages of liposomal-targeted drug, it is important to remove free drug and targeting agent from the final injectable suspension.
  • the liposomes in the suspension are pelleted by high-speed centrifugation leaving free compound and very small liposomes in the supernatant.
  • Another method involves concentrating the suspension by ultrafiltration, then resuspending the concentrated liposomes in a drug-free replacement medium.
  • gel filtration can be used to separate large liposome particles from solute molecules.
  • the liposome suspension is brought to a desired concentration for use in intravenous administration. This can involve resuspending the liposomes in a suitable volume of injection medium, where the liposomes have been concentrated, for example by centrifugation or ultraf ⁇ ltration, or concentrating the suspension, where the drug removal step has increased total suspension volume.
  • the suspension is then sterilized by filtration as described above.
  • the liposome-ligand preparation may be administered parenterally or locally in a dose which varies according to, e.g., the manner of administration, the drug being delivered, the particular disease being treated, etc.
  • the dose of the compound varies according to, e.g., the particular compound, the manner of administration, the particular disease being treated and its severity, the overall health and condition of the patient, and the judgment of the prescribing physician.
  • the dose of a contemplated SLe x analogue compound is in the range of about 50 ⁇ g to 10,000 mg/day for a 70 kg patient.
  • therapeutic administration should begin as soon as possible after the myocardial infarction or other injury.
  • a pharmaceutical composition is intended for parenteral, topical, oral or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment.
  • a pharmaceutical composition can be administered in a variety of unit dosage forms depending upon the method of administration.
  • administration include powder, tablets, pills, capsules and dragees.
  • a pharmaceutical composition is administered intravenously.
  • this invention provides a composition for intravenous administration that comprises a solution of a contemplated SLe x analogue compound dissolved or dispersed in a pharmaceutically acceptable diluent (carrier), preferably an aqueous carrier.
  • a pharmaceutically acceptable diluent carrier
  • aqueous carriers can be used, e.g., water, buffered water, 0.4 percent saline, and the like.
  • These compositions can be sterilized by conventional, well known sterilization techniques, or can be sterile filtered.
  • the resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • a composition can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • the concentration of SLe x analogue compound utilized is usually about 0.1 to about 10-30 mol % and is selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. As described above, the composition components can be delivered via liposome preparations.
  • a typical pharmaceutical composition for intravenous infusion can be made up to contain 250 ml of sterile Ringer's solution, and 25 mg of the SLe x analogue compound.
  • Actual methods for preparing parenterally administrable compounds are known or apparent to those skilled in the art and are described in more detail in for example, Remington's Pharmaceutical Sciences. 17th ed., Mack Publishing Company, Easton, PA (1985), which is incorporated herein by reference.
  • nontoxic solid diluents for solid compositions, conventional nontoxic solid diluents (carriers) may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95 percent of active ingredient, that is, a before-described SLe x analogue compound, preferably about 20 percent (see,
  • SLe x analogue compound is preferably supplied in finely divided form along with a surfactant and propellant.
  • Typical percentages of a SLe x analogue compound are about 0.5 to about 30 percent by weight, and preferably about 1 to about 10 percent.
  • the surfactant must of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing .from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropylene derivatives of these esters.
  • the surfactant can constitute about 0.1 to about 20 percent by weight of the composition, and preferably about 0.25 to about 5 percent.
  • the balance of the composition is ordinarily propellant.
  • Liquefied propellants are typically gases at ambient conditions, and are condensed under pressure.
  • suitable liquefied propellants are the lower alkanes containing up to 5 carbons, such as butane and propane; and preferably fluorinated or fluorochlorinated alkanes. Mixtures of the above can also be employed.
  • a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided compounds and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve.
  • a pharmaceutical composition containing a SLe x analogue compound can be administered for prophylactic and/or therapeutic treatments.
  • a pharmaceutical composition containing a SLe x analogue compound can be administered for prophylactic and/or therapeutic treatments.
  • a composition is administered to a patient already suffering from a disease, as described above, in an amount sufficient to inhibit binding between cells expressing a selectin and neutrophils or HL-60 cells; i.e., cure or at least partially arrest the symptoms of the disease and its complications.
  • An amount adequate to accomplish this is defined as “therapeutically effective dose” or "a cell adhesion-inhibiting amount”. Amounts effective for this use depend on the severity of the disease and the weight and general state of the patient, but generally range from about 0.5 mg to about 10,000 mg of SLe x analogue compound per day for a 70 kg patient, with dosages of from about 5 mg to about 2,000 mg of a compound per day being more commonly used.
  • a composition containing a contemplated compound is administered to a patient susceptible to or otherwise at risk of a particular disease.
  • Such an amount is defined to be a "prophylactically effective dose” and is also an amount sufficient to inhibit adhesion (binding) of SLe x -containing cells to selectin.
  • the precise amounts again depend on the patient's state of health and weight, but generally range from about 0.5 mg to about 5,000 mg per 70 kilogram patient, more commonly from about 5 mg to about 2,000 mg per 70 kg of body weight.
  • Another way to assess an adhesion-inhibiting amount of a contemplated SLe x analogue compound is to compare binding inhibition exhibited by the SLe x analogue compound to that provided by SLe x itself.
  • One convenient way to make that comparison is by use of IC 50 (the concentration needed to inhibit binding by one-half) of the two compared materials, and base the amount used on the amount of SLe x and an amount of the SLe x analogue compound that is a multiple of the IC 50 value for that compound.
  • a compound whose IC 50 value is about one-tenth that of SLe x itself, when used at ten times the molar amount of SLe x is a useful cell adhesion-inhibiting amount. More preferably, the amount is about four times the amount of SLe x . More preferably still, the amount is equal to that of SLe 1 . Most preferably, as is the case with most of the SLe x analogue compounds described herein, the amount used is less than the amount of SLe x used such as about one-half to about one-tenth the molar amount of SLe x itself.
  • Single or multiple administrations of a composition can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical formulations should provide a quantity of a SLe x analogue compound sufficient to effectively treat the patient.
  • the compounds can also find use as diagnostic reagents.
  • labeled compounds can be used to locate areas of inflammation or tumor metastasis in a patient suspected of having an inflammation.
  • the compounds can be labeled with 125 I, 14 C, or tritium.
  • This example provides a synthesis of N-benzyl lactosamine acetate salt (Compound 2).
  • This example provides the preparation of lactosamine acetate salt
  • Compound 2 from the previous reaction was equipped with a three-way stopcock and was put through an argon/vacuum/purge cycle three times using a balloon of argon and a house vacuum line.
  • the flask was opened and 10 percent palladium on carbon was added (7.4 gm, 6.98 mmol).
  • the flask was then re-equipped with a three-way stopcock and put through a vacuum/purge cycle three times using hydrogen gas.
  • the reaction was then held under a hydrogen atmosphere using a balloon.
  • the crude acidic methanolic solution of Compound 3 was diluted with 14 mL of H 2 O and treated with sodium carbonate (29.7 gm, 280 mmol) followed by phthalic anhydride (20.7 gm, 140 mmol). The reaction was watched carefully because some foaming occurs initially. After four hours, the reaction was complete, and the slurry was filtered through a sintered glass funnel to remove residual sodium carbonate and phthalate-based material. The filtrate was then concentrated to a paste first by rotary evaporation and then under high vacuum to provide Compound 4. Removing as much of the trace methanol and H 2 O left in the material is essential to avoid side reaction with acetic anhydride in the following acetylation.
  • the example provides the conversion of 1,3,6-tri-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl- ⁇ -D-galactopyranosyl)- ⁇ -D-glucopyranoside to Compound 5.
  • the crude concentrated product was dissolved in pyridine 50 mL and treated with acetic anhydride (25 mL). After stirring overnight (about 18 hours), TLC in 20: 1 CHCl 3 :MeOH indicated preponderance of one major UV active spot that cospotted with authentic Compound 5.
  • the solution was cooled to zero degrees C, treated with 7.5 mL of H 2 O, and stirred for 15 minutes to hydrolyze excess acetic anhydride.
  • the solution was diluted to 250 mL with dichloromethane and washed (3 ⁇ 250 mL) with 2N HCl, (3 ⁇ 250 mL) with saturated NaHCO 3 , and (1 ⁇ 250 mL) with saturated NaCl.
  • the organic solution was dried (MgSO 4 ), filtered, and concentrated to a crude product.
  • the crude product was then dissolved in a minimum of methanol and once again
  • This example provides an alternative preparation of Compound 5 from Lactuiose.
  • a 300 mL stainless steel autoclave containing a stirbar, lactuiose (17.1 g, 50 mmol), and ammonium acetate (3.85 g, 50 mmol) was cooled to -78 °C and charged with 80 mL of liquid ammonia.
  • the autoclave was sealed and allowed to warm to room temperature with stirring. Once the autoclave had reached room temperature, it was placed in an oil bath and heated to 35°C for 24 hours. The autoclave was then cooled to room temperature and carefully vented to the atmosphere. Once all of the ammonia had dissipated, approximately two hours, the entire autoclave was placed in a vacuum desiccator containing phosphorous pentoxide and carefully put under high vacuum.
  • Lactuiose aminoglycoside (Compound 10) (3.41 gm, 10 mmol) was dissolved in 100 mL of anhydrous methanol and stirred at room temperature under argon. Glacial acetic acid (572 uL, 10 mmol) was then added. After 24 hours, the yellow solution was concentrated to a foam that appeared to contain lactosamine acetate salt as a 1:1 mixture of ⁇ and ⁇ anomers. Two other products were apparent which are thought to be the ⁇ and ⁇ anomers of galactopyranosyl mannosamine. This product was used crude in the following reaction.
  • This example provides a synthesis of 1-Chloro-3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl- ⁇ -D-galactopyranosyl)- ⁇ -D-glycopyranoside (Compound 6).
  • EXAMPLE 8 This example provides the synthesis of Ethyl 4,6-O-benzylidene- ⁇ -D-galactopyranoside (Compound 8).
  • Ethyl ⁇ -D-galactopyranoside, Compound 7, (0.851 kg, 4.09 mol) was charged into a 20 L rotovap flask with toluene sulfonic acid (1.5 g, 7.9 mmol).
  • the evaporator flask was fixed to the evaporator and benzaldehyde dimethyl acetal (1.23 L, 8.18 mol) was added by aspiration.
  • the mixture was tumbled for four hours. Between thirty and forty minutes after addition of the acetal, near complete solution was obtained followed rapidly by the appearance of a heavy precipitate. Rotation was continued for four hours at which time triethylamine (1.5 mL) was added to neutralize the reaction mixture.
  • Ethyl 4,6-O-benzylidene- ⁇ -D-galactopyranoside, Compound 8, (0.924 kg, 3.12 mol) was put into a 20 liter reactor equipped with an air drive, a pressure equalizing addition funnel with gas inlet, cooling bath, and a gas outlet. Before sealing the flask, dichloromethane (9.3 L) and pyridine (2 L) were added, which gave a homogeneous solution. The addition funnel was charged with chloroacetyl chloride (0.388 kg, 3.43 mol, 273 mL) as a 60 percent solution in dichloromethane. The flask was sealed and a low flow of dry nitrogen was begun.
  • EXAMPLE 10 This example provides the preparation of Ethyl ( ⁇ -D-galactopyranosyl)-(1- 4)-O-(2-N-allyloxycarbonyl-2-deoxy- ⁇ -D-glucopyranosyl)-(1-3)-O- ⁇ -D-galactopyranoside Compound 10).
  • This example provides the preparation of Ethyl [sodium (5-acetamido-3,5-dideoxy- ⁇ -D-glycero-D-galacto-nonulopyranosylonate)]-(2-3)-O-( ⁇ -D-galactopyranosyl)-(1-4)-O-(2-N-allyloxycarbonyl-2-deoxy- ⁇ -D-glucopyranosyl)-(1-3)-O- ⁇ -D-galactopyranoside (Compound 11).
  • phosphoenolpyruvate (0.177 kg), potassium chloride (0.087 kg), sodium azide (8.4 g), and uridine-5'-diphosphate (8.76 g) were added and the resulting mixture stirred until all of the solids are dissolved.
  • a solution of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (0.528 kg) was prepared in water (15 L) and the pH of the resulting solution was adjusted to 7.5.
  • Bovine serum albumin (22 g), sodium azide (11.5 g), sialic acid (0.242 kg), phosphoenolpyruvate (0.395 kg), cytidine-5'-monophosphate (25 g), adenosine-5'-triphosphate (4.7 g), manganese chloride (1 M, 780 mL) are added.
  • This example provides the synthesis of Ethyl [methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto-nonulopyranosylonate)]-(2-3)-O-(2,4,6-tri-O-acetyl- ⁇ -D-galactopyranosyl)-(1-4)-O-(3,6-di-O-acetyl-2-N-allyloxycarbonyl-2-deoxy- ⁇ -D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl- ⁇ -D-galactopyranoside
  • the evaporation flask was charged with a solution of N,N- dimethylaminopyridine (20 g) in pyridine (12 L).
  • the rotavapor bath was charged with ice-water mixture, and rotation was continued while acetic anhydride (6 L) was added during a period of one hour. Two hours after complete addition, more acetic anhydride (2 L) was added and the resulting mixture was left for 20 hours rotating slowly at room temperature. To ensure compete acetylation, more acetic anhydride (1 L) was added and the mixture was rotated for an additional 24 hours.
  • the reaction was checked by TLC (ethyl acetate: hexane: ethanol, 10: 10:3).
  • This example provides the synthesis of Ethyl [methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto-2-nonulopyranosylonate]-(2,3)-O-(2,4,6-tri-O-acetyl- ⁇ -D-galactopyranosyl)-(1,4)-O-(2-amino-2-deoxy-3,6-di-O-acetyl- ⁇ -D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl- ⁇ -D-galactopyranoside (Compound 13).
  • This example provides the synthesis of Ethyl [methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto-2-nonulopyranosylonate]-(2,3)-O-(2,4,6-tri-O-acetyl-i8-D-galactopyranosyl)-(1,4)-O-(2-amino-2-deoxy-6-O-acetyl- ⁇ -D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl- ⁇ -D-galactopyranoside (Compound 14).
  • EXAMPLE 15 This example provides the synthesis of Ethyl [methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto-2-nonulopyranosylonate]-(2,3)-O-(2,4,6-tri-O-acetyl- ⁇ -D-galactopyranosyl)-(1,4)-O-(2-benzamido-2-deoxy-6-O-acetyl- ⁇ -D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl- ⁇ -D-galactopyranoside (Compound 15).
  • EXAMPLE 16 This example provides the sythesis of Ethyl [methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto-2-nonulopyranosylonate]-(2,3)-O-(2,4,6-tri-O-acetyl- ⁇ -D-galactopyranosyl)-(1,4)-O-[(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-(1,3)]-O-(2-benzamido-2-deoxy-3,6-di-O-acetyl- ⁇ -D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl- ⁇ -D-galactopyranoside (Compound 16).
  • This example provides the synthesis of Ethyl (5-acetamido-3,5-dideoxy- ⁇ -D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-( ⁇ -D-galactopyranosyl)-(1 ,4)-O-[( ⁇ -L-fucopyranosyl)-(1,3)]-O-(2-benzamido-2-deoxy- ⁇ -D-glucopyranosyl)-(1,3)-O- ⁇ -D-galactopyranoside (Compound 17).
  • This example provides the preparation of Ethyl [methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto-2-nonulopyranosylonate]-(2,3)-O-(2,4,6-tri-O-acetyl- ⁇ -D-galactopyranosyl)-(1,4)-O-[( ⁇ -L-fucopyranosyl)-(1,3)]-O-(2-2'-napthamido-2-deoxy-3,6-di-O-acetyl- ⁇ -D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl- ⁇ -D-galactopyranoside (Compound 29).
  • Tables 1, 2 and 3 show the generalized structures for groups of compounds corresponding to Compounds 15, 16 or 17, and provides other pertinent data for each of those compounds.
  • Table 1 shows the acylating agent used to prepare each R 1 group.
  • Tables 1-3 are followed by NMR and added data for several of those compounds, and inhibitor Compounds 30-38, including last step yields.
  • This example provides the preparation of Ethyl [methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto-2-nonulopyranosylonate]-(2,3)-O- (2,4,6-tri-O-acetyl- ⁇ -D-galactopyranosyl)-(1,4)-O-(2-benzyloxycarbonylamido-2-deoxy-6-O-acetyl- ⁇ -D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl- ⁇ -D-galactopyranoside
  • This example provides the preparation of Ethyl [methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto-2-nonulopyranosylonate]-(2,3)-O-(2,4,6-tri-O-acetyl- ⁇ -D-galactopyranosyl)-(1,4)-O-[(2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl)-(1,3)]-O-(2-benzyloxycarbonylamido-2-deoxy-3,6-di-O-acetyl- ⁇ -D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl- ⁇ -D-galactopyranoside (Compound 40).
  • This example provides the preparation of Ethyl [methyl (5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto-2-nonulopyranosylonate]-(2,3)-O- (2,4,6-tri-O-acetyl- ⁇ -D-galactopyranosyl)-(1,4)-O-[( ⁇ -L-fucopyranosyl)-(1,3)]-O-(2-amino-2-deoxy-3,6-di-O-acetyl- ⁇ -D-glucopyranosyl)-(1,3)-O-2,4,6-tri-O-acetyl- ⁇ -D-galactopyranoside (Compound 41).
  • This example illustrates the preparation of esterified forms of compound 17 beginning with Ethyl (sodium (5-acetamido-3,5-dideoxy- ⁇ -D-glycero-D-galacto-nonulopyranosylonate))-(2-3)-O-( ⁇ -D-galactopyranosyl)-(1-4)-O-(2-N-allyloxycarbonyl-2-deoxy- ⁇ -D-glucopyranosyl)-(1-3)-O- ⁇ -D-galactopyranoside (Compound 11)
  • the evaporation flask was charged with a solution of N,N- dimethylaminopyridine (20 g) in pyridine (12 L).
  • the rotavapor bath was charged with ice-water mixture, and rotation was continued while acetic anhydride (6 L) was added during a period of one hour. Two hours after complete addition, more acetic anhydride (2 L) was added and the resulting mixture was left for 20 hours rotating slowly at room temperature. To ensure compete acetylation, more acetic anhydride (1 L) was added and the mixture was rotated for an additional 24 hours.
  • the reaction was checked by TLC (ethyl acetate:hexane:ethanol, 10:10:3). Upon complete reaction, vacuum was applied and 14 L of distillate were collected.
  • Alcohol (1 L) is added to destroy excess acetic anhydride during which a slight exotherm is typically noticed. After 1 hour, the mixture is concentrated to a syrup, and transferred to a 50 L extractor with the aid of ethyl acetate-water mixture (13/13 L). The mixture is agitated vigorously. After phase separation, the lower aqueous layer is drawn off, and the remaining organic layer is filtered through paper. The filtrate is washed with 5 percent aqueous hydrochloric acid (15 L, the aqueous layer should still be strongly acidic to pH-paper after washing), and aqueous 1 M sodium bicarbonate (15 L, the aqueous layer should still be alkaline to pH paper after washing). The organic layer is then transferred to a 20 L container, dried over anhydrous sodium sulfate and filtered.
  • dichloromethane acetone (25 L), and finally with 1:1 dichloromethane: acetone (50 L) to give fractions containing product.
  • the fractions containing product are evaporated, and redissolved in dichloromethane (1.5 L). This solution is slowly added to a vigorously stirred mixture of ethyl ether (7.5 L) and hexane (10 L). The resulting precipitate is filtered and washed with 2:1 ether:hexane, air-dried overnight, then dried in high vacuum for 48 hours to provide a purified product of the ester-form of the blocked tetrasaccharide.
  • the resulting reaction mixture is diluted with acetic acid (600 mL) and washed 1 x 200 mL with H 2 O and 1 ⁇ 200 mL with saturated NaCl solution.
  • the organic solution is dried (MgSO 4 ), filtered, concentrated by rotary evaporation, and flash chromatographed on a 65 mm x 10" column of silica gel using 3:1 EtOAc:acetone as eluant.
  • the product-containing fractions (as judged by TLC) are pooled and
  • the product-containing fractions are pooled and concentrated by rotary evaporation and high vacuum.
  • To a stirred solution of the resulting product in 1 mL of dichloroethane at room temperature under an argon atmosphere are added powdered, flame-dried 4 ⁇ molecular sieves (100 mg), tetramethylurea (120 ⁇ L, 1 mmol), and tri-O-benzyl fucosyl fluoride (218 mg, 0.5 mmol).
  • the reaction is cooled to -20°C and treated with SnCl 2 (95 mg, 0.5 mmol) and AgClO 4 (126 mg, 0.5 mmol).
  • the reaction is then allowed to slowly warm to room temperature while stirring for 24 hours, or until TLC (10: 1 CHCl 3 :MeOH) shows the reaction is complete.
  • reaction mixture is filtered through a plug of celite with 50 mL of dichloromethane, and the filtrate is washed 2 ⁇ 50 mL with H 2 O.
  • organic solution is dried (MgSO 4 ), filtered, concentrated, and flash chromatographed on a 20 mm ⁇ 6" column of silica gel using 10: 10:3 EtOAc: hexane: MeOH as eluant.
  • the product-containing fractions (as judged by TLC) are pooled and concentrated by rotary
  • This oil is dissolved in 5 mL of an alcohol and stirred at room temperature in a capped flask. An alkoxide powder or pyridine is added to the stirred solution. The reaction mixture is stirred for 16 hours, or until TLC (60:50: 15 CHCl 3 :MeOH:15 mM CaCl 2 ) shows completion of the reaction. The mixture is treated with 1 gram of Dowex 50 ⁇ 8-400 cation exchange resin (hydrogen form, freshly alcohol washed) and stirred for one minute. The mixture is filtered through a fritted funnel and the filtrate concentrated by rotary evaporation to an oil.
  • Z is selected from the group consisting of hydrogen, C 1 -C 6 acyl and
  • Y is selected from the group consisting of C(O), SO 2 , HNC(O),
  • R 1 is selected from the group consisting of an aryl, a substituted aryl and a phenyl C 1 -C 3 alkylene group, wherein an aryl group has one five- membered aromatic ring, one six-membered aromatic ring or two fused six-membered aromatic rings, which rings are selected from the group consisting of hydrocarbyl,
  • a substituted aryl group is said aryl group having a substituent selected from the group consisting of a halo, trifluoromethyl, nitro, C 1 -C 12 alkyl, C 1 -C 12 alkoxy, amino, mono-C 1 -C 12 alkylamino, di-C 1 -C 12 alkylamino, benzylamino, C 1 -C 12 alkylbenzylamino, C 1 -C 12 thioalkyl and C 1 -C 12 alkyl carboxamido groups, or R 1 Y is allyloxycarbonyl or chloroacetyl; R 2 is selected from the group consisting of hydrogen, C 1 -C 18 straight chain, branched chain or cyclic hydrocarbyl, C 1 -C 6 alkyl C 1 -C 5 alkylene ⁇ -
  • R 4 is a member selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, benzyl, pentyl and hexyl.
  • R f 0.52 (3: 1 i-PrOH:NH 4 OAc), white solid, 35 mg, 96 percent.
  • Galactosyltransferase (240 u, 10u/mL) and uridine 5'-diphosphogalactose 4'-epimerase (330u, 10u/ ⁇ L) were added to a solution containing sodium cacodylate (pH 7.5, 1 M, 5.5 mL), water (45 mL), MnCl 2 (1 M, 1.4 mL), alkaline phosphatase (1000u, 1u/ ⁇ L), uridine diphosphoglucose disodium salt (3.86 g, 6.81 mmol), and ethyl 2-deoxy-2-allyloxycarboxamido- ⁇ -D-glucopyranoside (1.32 g, 4.54 mmol).
  • the reaction mixture was inverted several times and then allowed to sit at room temperature. After 2 days, the pH value was adjusted to 7.4 by adding 1 M NaOH (aq), and then additional galactosyltransferase (50u, 10u/mL) and uridine 5'-diphosphogalactose 4'-epimerase (50u, 10u/ ⁇ L) were added. The mixture was allowed to stand for another 2 days.
  • Ethyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto- 2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl- ⁇ -D-gaIactopyranyl)-(1-4)-O-3,6-di-O-acetyl-2-deoxy-2-amino- ⁇ -D-glucopyranoside (57).
  • Ethyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyI- ⁇ -D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl- ⁇ -D-galacto-pyranosyl)-(1-4)-O-[2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl-(1-3)-O]-6-O-acetyl-2-deoxy-2-(4-nitrobenzamido)- ⁇ -D-gluco-pyranoside (59).
  • Ethyl sodium 5-acetamido-3,5-dideoxy- ⁇ -D-glycero-D-galacto-2-nonulopyranuronate
  • Ethyl (methyl 5-aceta ⁇ nido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl- ⁇ -D-galactopyranosyI)-(1-4)-O-(6-O-acetyl-2-deoxy-2-(4-nitrobenzamido)- ⁇ -D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O- ⁇ -D-galactopyranoside (74).
  • Ethyl (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl- ⁇ -D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyI- ⁇ -D-galactopyranosyl)-(1-4)-O-[2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl-(1-3)-O]-(6-O-acetyl-2-deoxy-2-(4-nitrobenzamido)- ⁇ -D-glucopyranosyl)-(1-3)-O-2,4,6-tri-O- ⁇ -D-galactopyranoside (75).
  • Ethyl sodium 5-acetamido-3,5-dideoxy- ⁇ -D-glycero-D-galacto-2-nonulopyranuronate
  • 2-3)-O-( ⁇ -D-galactopyranosyl) -(1-4)-O-[2,3,4-tri-O-benzyI- ⁇ -L-fucopyranosyl-(1-3)-O]-[2-deoxy-2-(4-nitrobenzamido)- ⁇ -D-glucopyranosyl]-(1-3)-O- ⁇ -D-galactopyranoside (76).
  • Ethyl sodium 5-acetamido-3,5-dideoxy- ⁇ -D-glycero-D-galacto-2-nonulopyranuronate
  • Ethyl sodium 5-acetamido-3,5-dideoxy- ⁇ -D-glycero-D-galacto-2-nonulopyranuronate
  • the mixture was purged three times with hydrogen gas and then stirred under an atmosphere of hydrogen for 3 days. After being degassed, the mixture was then filtered through celite with washing with MeOH. The filtrate was concentrated and dried in vacuo. The residue was dissolved in 10 mL of pyridine, and the solution was cooled to 0°C. Three mL of Ac 2 O was added and stirring was continued at 0°C for 30 min, and then at room temperature for 2 days at which time TLC showed acetylation was complete. The reaction mixture was diluted with EtOAc, and washed with ice-water, sat. NaHCO 3 (aq) and brine. The organic layer was dried over Na 2 SO 4 , filtered and concentrated. Purification by flash chromatography on silica gel using
  • Trichloroacetimido (methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-of-D-glycero-D-galacto-2-nonulopyranuronate)-(2-3)-O-(2,4,6-tri-O-acetyl- ⁇ -D-galactopyranosyl)-(1-4)-O-[2,3,4-tri-O-acetyl- ⁇ -L-fucopyranosyl-(1-3)-O]-(6-O-acetyl-2-deoxy-2-acetamido- ⁇ -D-gIucopyranosyl)-(1-3)-O-2,4,6-tri-O-acetyl- ⁇ -D-galactopyranoside (82).
  • Benzoyl chloride (9.09 g, 64.7 mmol, 7.51 mL) was then added dropwise at -65° over 10 min. The stirring was continued for 2 h at -65°C and then at room temperature for 18 h. The reaction mixture was then washed with 1N HCl (400 mL), water (250 mL ⁇ 2), saturated NaHCO 3 solution (500 mL). The aqueous layers were extracted with EtOAc/EtOH (8:2) and the combined organic layers were dried (MgSO 4 ) and
  • Amino (ammonium 5-acetamido-3,5-dideoxy- ⁇ -D-glycero-D-galacto-2-nonulopyranosylonate)-(2,3)-O-( ⁇ -D-galactopyranosyl)-(1,4)-O-[ ⁇ -L-fucopyranosyl- (1,3)-O]-(2-acetamido-2-deoxy- ⁇ -D-glucopyranosyl)-(1,3)-O- ⁇ -D-galactopyranoside
  • Bromoacetic anhydride (0.31 g, 1.195 mmoles) was added to a solution of 99 (0.1 g, 0.0996 mmol) containing ethanol (2 mL), water (2 mL) and sat. NaHCO 3 (2 mL).
  • the mixture was inverted several times, and then allowed to sit at room temperature for 2 days.
  • the pH was adjusted to 7.4 by addition of 1 M NaOH solution, and then additional enzymes (50% amounts of first addition) were added.
  • the mixture was allowed to stand for 5 days at which time the starting material had been consumed.
  • potassium 2-phosphoenol pyruvate (3.75 g)
  • cytidine monophosphate (289 mg)
  • ATP 100 mg
  • sialic acid 4.1g
  • sodium cacodylate pH 7.4, 1 M, 40 mL
  • the mixture was purged three times with hydrogen gas and then stirred under hydrogen atmosphere (1atm) for 1 h.
  • the mixture was degassed and concentrated. 10 mL of MeOH was added, and the mixture was filtered through celite with washing with MeOH. The filtrate was concentrated and dried in vacuo overnight to provide amine which was used for the next reaction.
  • dimyristoylphosphatidyl thioethanol 2.0 mg (6.13 ⁇ mol) of Cs 2 CO 3 and 0.6 mL of DMF was stirred under argon at room temperature for 2.5 h. The solvent was removed by co-evaporation with water under the reduced pressure. The residue obtained was purified by chromatography on reverse phase silica (Biosil C-18; 50-85% MeOH in H 2 O) to afford the liposomal pentasaccharide 115 as a white solid after lyophilization (4.0 mg, 55%).
  • This example provides a general procedure for coupling of a lipid to compounds 62, 73, 78, 85 and 100 as provided in Figure 7.

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Abstract

La présente invention se rapporte à des analogues de sialyle Lewisx (Lex) qui inhibent l'adhésion cellulaire entre une sélectine et des cellules qui expriment le sialyle Lex au niveau de leurs surfaces, ainsi qu'à des procédés et des compositions utilisant ces analogues, des intermédiaires et des procédés de préparation de composés inhibiteurs de l'adhésion cellulaire et de leurs intermédiaires. Un composé intermédiaire ou inhibiteur particulièrement considéré est représenté par la formule (A), dans laquelle Z est sélectionné dans le groupe constitué par l'hydrogène, un acyle C¿1?-C6 et le composé représenté par la formule (B), Y est sélectionné dans le groupe constitué par C(O), SO2, HNC(O), OC(O) et SC(O), R?1¿ est sélectionné dans le groupe constitué par un groupe se liant à un lipide, un lipide, un groupe de liaison comportant un lipide fixé, un aryle, un aryle substitué et un groupe phényle alkylène C¿1?-C3, où un groupe aryle possède un noyau aromatique à cinq ou six éléments, un noyau aromatique condensé à cinq ou six éléments, ou deux noyaux aromatiques condensés à six éléments, lesdits noyaux étant sélectionnés dans le groupe constitué par des noyaux hydrocarbyle, monooxahydrocarbyle, monothiahydrocarbyle, monoazahydrocarbyle et diazahydrocarbyle, et un groupe aryle substitué est ledit groupe aryle ayant un substituant sélectionné dans le groupe constitué par un groupe halo, trifluorométhyle, nitro, alkyle C1-C18, alcoxy C1-C18, amino, mono-alkylamino C1-C18, di-alkylamino C1-C18, benzylamino, alkylbenzylamino C1-C18, thioalkyle C1-C18 et alkyle C1-C18 carboxamido, ou bien R?1¿Y est allyloxycarbonyle ou chloroacétyle, R2 est sélectionné dans le groupe constitué par l'hydrogène, un groupe se liant à un lipide, un lipide, un groupe de liaison comportant un lipide fixé, un hydrocarbyle cyclique, à chaîne droite ou ramifiée C¿1?-C18, un alkyle C1-C6 alkylène C1-C5 φ-carboxylate, φ-tri(alkyl C1-C4/phényl)silyle alkylène C2-C4, monosaccharide et disaccharide, ou bien OR?2¿ forme un hydrocarbyle carbamate cyclique, à chaîne droite ou ramifiée C¿1?-C18, R?3¿ est hydrogène ou acyle C¿1?-C6, R?4¿ est hydrogène, alkyle C¿1?-C6 ou benzyle, R?5¿ est sélectionné dans le groupe constitué par l'hydrogène, un groupe benzyle, méthoxybenzyle, diméthoxybenzyle et acyle C¿1?-C6, R?7¿ est méthyle ou hydroxyméthyle et X est sélectionné dans le groupe constitué par acyloxy C¿1?-C6, hydroxylacyloxy C2-C6, hydroxy, halo et azido.
PCT/US1996/016559 1995-10-18 1996-10-16 Analogues de sialyle lewisx utilises en tant qu'inhibiteurs de l'adhesion cellulaire WO1997014707A1 (fr)

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US7060685B2 (en) 2002-05-16 2006-06-13 Glycomimetics, Inc. Compounds and methods for inhibiting selectin-mediated function
US7361644B2 (en) 2003-11-19 2008-04-22 Glycomimetics, Inc. Specific antagonist for both E- and P-selectins
US7517980B2 (en) 2005-08-09 2009-04-14 Glycomimetics, Inc. Glycomimetric inhibitors of the PA-IL lectin, PA-IIL lectin or both the lectins from Pseudomonas
USRE44778E1 (en) 2005-09-02 2014-02-25 Glycomimetics, Inc. Heterobifunctional pan-selectin inhibitors
US8895510B2 (en) 2008-04-08 2014-11-25 Glycomimetics, Inc. Pan-selectin inhibitor with enhanced pharmacokinetic activity
US8921328B2 (en) 2010-09-14 2014-12-30 Glycomimetics, Inc. E-selectin antagonists
US9109002B2 (en) 2011-12-22 2015-08-18 Glycomimetics, Inc. E-selectin antagonist compounds, compositions, and methods of use
US9867841B2 (en) 2012-12-07 2018-01-16 Glycomimetics, Inc. Compounds, compositions and methods using E-selectin antagonists for mobilization of hematopoietic cells
US10519181B2 (en) 2014-12-03 2019-12-31 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectins and CXCR4 chemokine receptors
US11045485B2 (en) 2016-01-22 2021-06-29 Glycomimetics, Inc. Glycomimetic inhibitors of PA-IL and PA-IIL lectins
US11072625B2 (en) 2016-10-07 2021-07-27 Glycomimetics, Inc. Highly potent multimeric e-selectin antagonists
US11197877B2 (en) 2017-03-15 2021-12-14 Glycomimetics. Inc. Galactopyranosyl-cyclohexyl derivauves as E-selectin antagonists
US11291678B2 (en) 2016-03-02 2022-04-05 Glycomimetics, Inc Methods for the treatment and/or prevention of cardiovascular disease by inhibition of E-selectin
US11433086B2 (en) 2016-08-08 2022-09-06 Glycomimetics, Inc. Combination of T-cell checkpoint inhibitors with inhibitors of e-selectin or CXCR4, or with heterobifunctional inhibitors of both E-selectin and CXCR4
CN115381844A (zh) * 2021-09-29 2022-11-25 北京大学 一种细菌荚膜寡糖衍生物及其制备方法、药物组合物和用途
US11548908B2 (en) 2017-12-29 2023-01-10 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3
WO2023062386A1 (fr) * 2021-10-14 2023-04-20 Owlstone Medical Limited Procédé de synthèse de sondes evoc
US11707474B2 (en) 2018-03-05 2023-07-25 Glycomimetics, Inc. Methods for treating acute myeloid leukemia and related conditions
US11712446B2 (en) 2017-11-30 2023-08-01 Glycomimetics, Inc. Methods of mobilizing marrow infiltrating lymphocytes and uses thereof
US11845771B2 (en) 2018-12-27 2023-12-19 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3

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Cited By (29)

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US7060685B2 (en) 2002-05-16 2006-06-13 Glycomimetics, Inc. Compounds and methods for inhibiting selectin-mediated function
US7361644B2 (en) 2003-11-19 2008-04-22 Glycomimetics, Inc. Specific antagonist for both E- and P-selectins
US7517980B2 (en) 2005-08-09 2009-04-14 Glycomimetics, Inc. Glycomimetric inhibitors of the PA-IL lectin, PA-IIL lectin or both the lectins from Pseudomonas
USRE44778E1 (en) 2005-09-02 2014-02-25 Glycomimetics, Inc. Heterobifunctional pan-selectin inhibitors
US9534009B2 (en) 2008-04-08 2017-01-03 Glycomimetics, Inc. Pan-selectin inhibitor with enhanced pharmacokinetic activity
US8895510B2 (en) 2008-04-08 2014-11-25 Glycomimetics, Inc. Pan-selectin inhibitor with enhanced pharmacokinetic activity
US8921328B2 (en) 2010-09-14 2014-12-30 Glycomimetics, Inc. E-selectin antagonists
US9796745B2 (en) 2011-12-22 2017-10-24 Glycomimetics, Inc. E-selectin antagonist compounds, compositions, and methods of use
US10526361B2 (en) 2011-12-22 2020-01-07 Glycomimetics, Inc. E-selectin antagonist compounds, compositions, and methods of use
US10766916B2 (en) 2011-12-22 2020-09-08 Glycomimetics, Inc. E-selectin antagonist compounds, compositions, and methods of use
US9109002B2 (en) 2011-12-22 2015-08-18 Glycomimetics, Inc. E-selectin antagonist compounds, compositions, and methods of use
US11987598B2 (en) 2011-12-22 2024-05-21 Glycomimetics, Inc. E-selectin antagonist compounds, compositions, and methods of use
US11332491B2 (en) 2011-12-22 2022-05-17 Glycomimetics, Inc. E-selectin antagonist compounds, compositions, and methods of use
US9867841B2 (en) 2012-12-07 2018-01-16 Glycomimetics, Inc. Compounds, compositions and methods using E-selectin antagonists for mobilization of hematopoietic cells
US10519181B2 (en) 2014-12-03 2019-12-31 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectins and CXCR4 chemokine receptors
US11045485B2 (en) 2016-01-22 2021-06-29 Glycomimetics, Inc. Glycomimetic inhibitors of PA-IL and PA-IIL lectins
US11291678B2 (en) 2016-03-02 2022-04-05 Glycomimetics, Inc Methods for the treatment and/or prevention of cardiovascular disease by inhibition of E-selectin
US11433086B2 (en) 2016-08-08 2022-09-06 Glycomimetics, Inc. Combination of T-cell checkpoint inhibitors with inhibitors of e-selectin or CXCR4, or with heterobifunctional inhibitors of both E-selectin and CXCR4
US11072625B2 (en) 2016-10-07 2021-07-27 Glycomimetics, Inc. Highly potent multimeric e-selectin antagonists
US11780873B2 (en) 2016-10-07 2023-10-10 Glycomimetics, Inc. Highly potent multimeric e-selectin antagonists
US11878026B2 (en) 2017-03-15 2024-01-23 Glycomimetics, Inc. Galactopyranosyl-cyclohexyl derivatives as e-selectin antagonists
US11197877B2 (en) 2017-03-15 2021-12-14 Glycomimetics. Inc. Galactopyranosyl-cyclohexyl derivauves as E-selectin antagonists
US11712446B2 (en) 2017-11-30 2023-08-01 Glycomimetics, Inc. Methods of mobilizing marrow infiltrating lymphocytes and uses thereof
US11548908B2 (en) 2017-12-29 2023-01-10 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3
US11707474B2 (en) 2018-03-05 2023-07-25 Glycomimetics, Inc. Methods for treating acute myeloid leukemia and related conditions
US11845771B2 (en) 2018-12-27 2023-12-19 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3
CN115381844A (zh) * 2021-09-29 2022-11-25 北京大学 一种细菌荚膜寡糖衍生物及其制备方法、药物组合物和用途
CN115381844B (zh) * 2021-09-29 2024-03-22 北京大学 一种细菌荚膜寡糖衍生物及其制备方法、药物组合物和用途
WO2023062386A1 (fr) * 2021-10-14 2023-04-20 Owlstone Medical Limited Procédé de synthèse de sondes evoc

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