EP0648223A1 - Derives de lactose substitues utilises comme inhibiteurs d'adhesion cellulaire - Google Patents

Derives de lactose substitues utilises comme inhibiteurs d'adhesion cellulaire

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Publication number
EP0648223A1
EP0648223A1 EP93916790A EP93916790A EP0648223A1 EP 0648223 A1 EP0648223 A1 EP 0648223A1 EP 93916790 A EP93916790 A EP 93916790A EP 93916790 A EP93916790 A EP 93916790A EP 0648223 A1 EP0648223 A1 EP 0648223A1
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EP
European Patent Office
Prior art keywords
compound
selectin
benzoyl
benzyl
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93916790A
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German (de)
English (en)
Other versions
EP0648223A4 (fr
Inventor
Saeed Abbas
Falguni Dasgupta
Darwin Asa
John H. Musser
Mina Chemistry Dep./Science Facility Nashed
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Glycomed Inc
Original Assignee
Glycomed Inc
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Application filed by Glycomed Inc filed Critical Glycomed Inc
Publication of EP0648223A1 publication Critical patent/EP0648223A1/fr
Publication of EP0648223A4 publication Critical patent/EP0648223A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
    • 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
    • C07H3/04—Disaccharides
    • 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 invention relates to compounds useful in the treatment of inflammation, allergic reactions, autoimmune diseases, and related conditions. More specifically, the invention concerns substituted lactose that binds to selectin receptors and to pharmaceutical compositions containing them. The present invention is also directed to synthetic methods useful in obtaining these analogs and other lactose derivatives.
  • receptor/ligand interactions are at least in part mediated by receptor/ligand interactions.
  • One class of receptors is known to recognize the peptide sequence "RGD"; other receptors recognize carbohydrate ligands.
  • L-SELECTIN L-SELECTIN
  • E-SELECTIN is perhaps the best characterized of the three selectins. It is particularly interesting because of its transient expression on endothelial cells in response to IL-1 or TNF (Bevilacqua, M.P., et al., Science (1989) 243:1160). The time course of this induced expression (2-8 hours) suggests a role for this receptor in initial neutrophil extravasation in response to infection and injury. Furthermore, Bevilacqua et al. (see Bevilacqua, M.P., et al., Proc. Natl. Acad. Sci.
  • L-SELECTIN appears to bind a sialic acid bearing Ugand based on neuraminidase treatment of peripheral lymph node high endothelial venules which inhibits L-SELECTIN recognition.
  • soluble L-SELECTIN in direct binding/inhibition assays suggests that certain carbohydrate moieties may be important ligand components including mannose and fucose, particularly when sulfated or phosphorylated. Imai et al., 1990 J. Cell Biol. Il l, 1225-1232. More recent studies suggest that L-Selectin binds to sialyl Lewis X. Foxall, C, et al., (1992) Cell, in press.
  • the ligand to P-SELECTIN is thought to have an epitope related to sialyl Lewis x. This conclusion is based on studies using antibody with this specificity that block P-SELECTIN mediated adhesion of HL-60 cells to activated platelets or COS cells that express P-SELECTIN. Larsen et al. (1990) Cell 63, 467-474. Other experiments have shown that the adhesion of HL-60 cells to P-SELECTIN transfected cells is blocked by the pentasaccharide isolated from milk that has the Lewis" epitope. Recently, P-Selectin has been shown to bind to sulfatides. Aruffo,
  • the invention provides agonists and antagonists which bind to selectin receptors and thus modulate the course of inflammation, cancer and related responses by modulating cell-cell adhesion events.
  • the invention is directed to compounds of the formula:
  • each R 1 is independently H or lower alkyl (1-4C);
  • R 2 is H, lower alkyl(l-4C), alkylaryl or one or more additional saccharide residues;
  • R 3 is a negatively charged moiety including SO4 ' , PO 4 ⁇ , or related group;
  • Y is H, OH 1 or lower alkyl(l-4C);
  • X is -CHR ⁇ CHOR ⁇ Cffi ⁇ OR 1 wherein R 4 and R 5 are each independently H, lower alkyl(l-4C), or taken together result in a five- or six-membered ring optionally containing a heteroatom selected from the group consisting of O, S, and NR 1 ; said five- or six-membered ring optionally substituted with one substituent selected from the group consisting of R ⁇ CH 2 OR ⁇ OR 1 , OOCR 1 , NR 1 NHCOR 1 , and SR 1 with the proviso that if X represents a hexose substituent R 4 and R 5 , taken together, cannot provide a hexose substituent.
  • the invention is directed to a method to synthesize lactose derivatives which method comprises contacting an intermediate of the formula
  • each R 6 is independently H, lower alkyl (1-4C), or a protecting group; and wherein Y 1 is H, OH 6 , OOCR 6 , or SR 6 ; wherein at least one R 6 , which is at the position to be substituted, and at most one adjacent R 6 is H and all other R 6 s are protecting groups; and R 7 is a protecting group, with an electrophile-donating moiety to obtain a product wherein the electrophile is substituted for the H of the OH at the position to be substituted.
  • the invention is directed to pharmaceutical compositions containing the compounds of formula 1 and to methods of treating inflammation using these compositions.
  • the invention is directed to compounds of formula 2 and additional intermediates in the synthesis of selectin binding ligands or other useful lactosyl residue-containing moieties.
  • the invention provides compounds that are useful in the treatment of inflammation by virtue of their ability to bind to selectin receptors. For example,
  • Figure 1 shows a diagrammatic view of the role believed to be played by one of the selectin receptors, ELAM-1, in mediating inflammation. Blood vessels are lined with endothelial cells capable of producing the ELAM-1 surface receptor.
  • Lymphocytes circulating in the vessel contain on their surfaces carbohydrate ligands capable of binding to the ELAM-1 receptor. This results in transfer of the lymphocyte through the vessel wall and into the surrounding tissue. While this may have a useful effect in some circumstances, as in cases when the surrounding tissue is infected, excessive transfer of the lymphocytes through the vessel wall and into the tissue may also be excessive and cause unwanted inflammation. While not wishing to be limited by any particular theory, it is believed that the compounds of the present invention which bind the ELAM-1 receptor, antagonize the action of the surface ligands on the circulating lymphocytes and thus prevent their transfer through the blood vessel wall to cause inflammation in the surrounding tissue.
  • assays for identifying lactose derivatives that act as selectin ligands involve contacting the appropriate selectin, L-SELECTIN, E- SELECTIN, or P-SELECTIN, with a putative ligand and measuring its binding properties.
  • both the selectin and the putative ligand may be in solution for a time sufficient for a complex to form consisting of the selectin and ligand , followed by separating the complex from uncomplexed selectin and ligand, and measuring the amount of complex formed.
  • the amount of uncomplexed selectin or compound could be measured.
  • a second and preferred assay format consist of immobilizing either the selectin or the putative ligand on a solid surface, and forming the selectin-ligand complex thereon by contacting the immobilized reagent with the non-immobilized reagent.
  • the selectin-ligand complex is separated from uncomplexed reagents, and the amount of complex formed can be determined by measuring the amount of the non-immobilized reagent present in the complex.
  • the putative ligand can be affixed to a microtiter well, followed by adding the desired selectin to the well and measuring the amount of selectin bound to the ligand.
  • a variation of the above assay is to genetically engineer cells to express high levels of L-SELECTIN, E-SELECTIN, or P-SELECTIN on their surface, and to use the cells in lieu of purified selectin.
  • Radiolabeled COS cells have been used in this type of assay, and can be transfected with cDNA that encodes for L- SELECTIN, E-SELECTIN or P-SELECTIN. After the cells have had a sufficient time to adhere to the ligand coated microtiter well, non-adherent cells are removed and the number of adherent cells determined. The number of adherent cells reflects the capacity of the ligand to bind to the selectin.
  • E-SELECTIN ligands For example, a complete cDNA for the ELAM-1 receptor was obtained by PCR starting with total RNA isolated from IL-1 stimulated human umbilical vein endothelium. The resulting cDNA was inserted into the CDM8 plasmid (see Aruffo, A., and Seed, B., Proc. Natl. Acad. Sci. USA (1987) 84:8573) and the plasmid amplified in coli. Plasmid DNA from individual colonies was isolated and used to transfect COS cells. Positive plasmids were selected by their ability to generate COS cells that support HL-60 cell adhesion. DNA sequencing positively identified one of these clones as encoding for ELAM-1 (Bevilacqua,
  • a full length cDNA encoding ELAM-1 was obtained by 35 cycles of the polymerase chain reaction with 1 ⁇ g of total RNA extracted from IL-1 stimulated human umbilical vein endothelial cells, utilizing primers complementary to the untranslated flanking sequences (5'-GGTGCGGCCGCGGCCAGAGACCCGAGGAGAG-3' and 5'-GGTGTCGACCCCACCTGAGAGATCCTG-3').
  • the 2Kb insert generated was gel purified, directionally cloned into the mammalian expression vector, CDM8 that had been modified by the insertion of a Sail site into the polylinker, and grown in E. coli (MC1061/p3). Plasmids were isolated from individual colonies and used to transfect COS cells.
  • Putative E-SELECTIN encoding plasmids were selected based on the ability of these transfected COS cells to support HL-60 cell adhesion 72 h posttransfection.
  • a positive cDNA whose sequence corresponded to the published sequence of E-SELECTIN with two nucleic acid substitutions was used in all experiments.
  • COS cells were transfected with 1 ⁇ g of this plasmid DNA per 3.5 - 5.0 x 10 5 cells, with 400 ⁇ g/ml DEAE-dextran and 100 ⁇ M chloroquine for 4 h, followed by a brief exposure to 10% DMSO in PBS. Cells were metabolically radiolabeled overnight with carrier free 32 PO 4 and harvested in PBS supplemented with 0.02% azide and 2 mM EDTA at 72 h posttransfection for use in cell adhesion studies.
  • E-SELECTIN transfected COS cells produced by the above method may be used to assay for glucuronyl glycolipid ligands.
  • COS cells may be transfected with cDNAs that encode L-SELECTIN and/or P-SELECTIN.
  • L-SELECTIN IgG chimera constructs have been previously described by Watson S. R. et al., (1990) J. Cell Biol. 110: 2221-2229. This chimera contains two complement binding domains, consistent with its natural expression. See Watson S. R. et al., (1991) J. Cell Biol. 115:235-243.
  • P-SELECTIN chimera was constructed in a similar manner as described by Walz, G., et al (1990) Science 250, 1132-1135, and Aruffo, A. et al.(1991) Cell, 67, 35-
  • any candidate compound of the formula 1 may be verified to bind ELAM-1 receptors by a positive result in the foregoing assays.
  • the compounds of formula 1 are useful in diagnostic and preparatory procedures both in vitro and in vivo.
  • Compounds of formula 1 may be conjugated to solid substrates and used for the purification of selectin receptor protein from biological samples. This is conducted most conveniently by arranging the coupled substrate as an affinity chromatography column and applying a sample putatively containing the selectin receptor protein to the affinity column under conditions wherein the selectin receptor protein is adsorbed whereas contaminating materials are not. The selectin receptor protein is then subsequently eluted, for example, by adjusting the eluent solution to containing competing amounts of the compound of formula 1 or by adjusting pH or salt parameters. Techniques for affinity purification are well understood, and routine optimization experiments will generate the appropriate conditions for conduct of the procedure.
  • the compounds of formula 1 are also useful as detection reagents to determine the presence or absence of selectin or related carbohydrate-binding receptor ligands.
  • a biological sample suspected to contain selectin receptor protein or a receptor protein closely related thereto is treated with the compound of formula 1 under conditions wherein complexation occurs between the receptor protein and the formula 1 compound, and the formation of the complex is detected.
  • a wide variety of protocols may be utilized in such procedures, analogous to protocols applied in immunoassays. Thus, direct assay wherein the amount of complex formed is directly measured may be utilized; alternatively, competition assays may be used wherein labeled selectin receptor protein is supplied along with, and in competition with, the biological sample.
  • the compounds of formula 1 in labeled form so that the complex is detected directly; in alternate procedures, the complex may be detected by size separations, secondary labeling reagents, or other alternate means.
  • Suitable labels are known in the art, and include radioactive labels, fluorescent labels, enzyme labels, chromogenic labels, or composites of these approaches.
  • the compounds of formula 1 may also be used as competitive diagnostic reagents to detect the quantity of selectin receptor-binding components, such as surface ligands, in biological fluids.
  • the compounds of formula 1 are labeled as described above and mixed with the biological sample and contacted with the appropriate receptor protein; the diminution of binding of the labeled compound of formula 1 to selectin receptor in the presence of biological sample is then determined.
  • the compounds of formula 1 may also be used in imagining studies in vivo to determine the location of selectin receptors in situ.
  • the compounds of formula 1 are supplied with labels which can be detected by in vivo imaging techniques, such as scintigraphic labels including indium 111, technetium 99, iodine 131, and the like.
  • Antibodies may also be prepared to the compounds of formula 1 by coupling these compounds to suitable carriers and administering the coupled materials to mammalian or other vertebrate subjects in standard immunization protocols with proper inclusion of adjuvants.
  • suitable immunogenic carriers include, for example, Keyhole Limpet Hemocyanin (KLH), tetanus toxoid, various serum albumins such as bovine serum albumin (BSA) and certain viral proteins such as rotaviral VP6 protein.
  • KLH Keyhole Limpet Hemocyanin
  • BSA bovine serum albumin
  • viral proteins such as rotaviral VP6 protein
  • the resulting antisera may be used per se or the antibody-secreting cells generated by the immunization may be immortalized using standard techniques and used as a source of monoclonal preparations which are immunoreactive with the compounds of formula 1.
  • the resulting antibodies are useful in assay systems for determining the presence and/or amount of the relevant formula 1 compound. Such assays are useful in monitoring the circulating levels of compounds of formula 1 in therapeutic treatments such as those described below.
  • the compounds of the invention are administered to a subject in need thereof for prophylactically preventing inflammation or relieving it after it has begun.
  • Treating as used herein means preventing or ameliorating inflammation and/or symptoms associated with inflammation.
  • the compounds are preferably administered with a pharmaceutically acceptable carrier, the nature of the carrier differing with the mode of administration, for example, oral administration, usually using a solid carrier and I.V. administration using a liquid salt solution carrier.
  • injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the compounds may also be emulsified or the active ingredient encapsulated in liposome vehicles.
  • Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
  • auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
  • Formulations may employ a variety of excipients including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin cellulose, magnesium carbonate, and the like.
  • Oral compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders. Particularly useful is the administration of the subject ligand molecules directly in transdermal formulations with permeation enhancers such as DMSO. Other topical formulations can be administered to treat dermal inflammation. In addition, transmucosal administration may be effected using penetrants such as bile salts or fusidic acid derivatives optionally in combination with additional detergent molecules. These formulations are useful in the preparation of suppositories, for example, or nasal sprays.
  • the vehicle composition will include traditional binders and carriers, such as polyalkylene glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about
  • Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function.
  • Diluents such as water, aqueous saline or other known substances can be employed with the subject invention.
  • the nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride.
  • a surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
  • compositions of the instant invention will contain from less than 1% to about 95% of the active ingredient, preferably about 10% to about 50%.
  • the active ingredient preferably about 10% to about 50%.
  • between about 10 mg and 50 mg will be administered to a child and between about 50 mg and 1000 mg will be administered to an adult.
  • the frequency of administration will be determined by the care given based on patient responsiveness.
  • Other effective dosages can be readily determined by one of ordinary skill in the art through routine trials establishing dose response curves.
  • it will be noted that it may not be desirable to completely block all selectin receptors of a particular type.
  • at least some of the white blood cells or neutrophils must be brought into the tissue in the areas where any wound, infection or disease state is occurring.
  • the amount of the selectin ligands administered as blocking agents must be adjusted carefully based on the particular needs of the patient while taking into consideration a variety of factors such as the type of disease that is being treated.
  • the compounds of the present invention are useful to treat a wide range of diseases, for example autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.
  • the compositions of the invention are applicable to treat any disease state wherein the immune system turns against the body causing the white cells to accumulate in the tissues to the extent that they cause tissue damage, swelling, inflammation and/or pain.
  • Formulations of the present invention might also be administered to prevent the undesirable after effects of tissue damage resulting from heart attacks.
  • a heart attack occurs and the patient has been revived, such as by the application of anticoagulants or thrombolytic (e.g., tPA)
  • thrombolytic e.g., tPA
  • the endothelial lining where a clot was formed has often suffered damage.
  • the antithrombotic has removed the clot
  • the damaged tissue beneath the clot and other damaged tissue in the endothelial lining which has been deprived of oxygen become activated.
  • the activated endothelial cells then synthesize selectin receptors, for example ELAM-1 receptors, within hours of the cells being damaged.
  • the receptors are extended into the blood vessels where they adhere to glycolipid ligand molecules on the surface of white blood cells. Large numbers of white blood cells are quickly captured and brought into the tissue surrounding the area of activated endothelial cells, resulting in inflammation, swelling and necrosis which
  • formulations of the invention In addition to treating patients suffering from the trauma resulting from heart attack, patients suffering from actual physical trauma could be treated with formulations of the invention in order to relieve the amount of inflammation and swelling which normally result after an area of the body is subjected to severe trauma.
  • Other conditions treatable using formulations of the invention include various types of arthritis and adult respiratory distress syndrome.
  • the compounds of formula 2 are intermediates in the preparation of compounds which contain a lactosyl unit.
  • the compounds of formula 2 are useful in the preparation of compounds of formula 1 whose use is described hereinabove.
  • alternative compounds containing a lactose residue may also be prepared, such as:
  • the affinity of the ligands of the invention for receptor can be enhanced by providing multiple copies of the ligand in close proximity, preferably using a scaffolding provided by a carrier moiety. It has been shown that provision of such multiple valence with optimal spacing between the moieties dramatically improves binding to receptor. For example, Lee, R.
  • the multivalency and spacing can be controlled by selection of a suitable carrier moiety.
  • Such moieties include molecular supports which contain a multiplicity of functional groups that can be reacted with functional groups associated with the ligands of the invention.
  • a particularly preferred approach involves coupling of the lactose-derived ligands of the invention to amino groups of the carrier through reductive amination.
  • Reductive animation is a particularly convenient way to couple aldehyde moieties to free amino groups by first forming the Schiff base and then treating the conjugate with a reducing agent, such as a hydride reducing agent.
  • the amino group-bearing carrier is mixed with the carbohydrate moiety at about pH 9 and allowed to form the Schiff base; the solvents are typically evaporated and reducing agent is added at high pH to complete the, reaction.
  • ligands include proteins and peptides, particularly those containing lysyl residues which have ⁇ -amino groups available for binding. It is also useful to include in the peptide or protein at least one tyrosine residue, as this offers a convenient site for labeling, for example with radioactive iodine.
  • a particularly convenient carrier to obtain a trivalent couple is the peptide Lys-Tyr-Lys. Complete reaction of the ligands of the invention with the free amino groups on this peptide result in a trivalent moiety.
  • X, Y, and R 1 , and R 3 are as above defined illustrate the multivalent compounds of the invention.
  • carriers including proteins such as BSA or HSA, a multiplicity of peptides including, for example, pentapeptides, decapeptides, pentadecapeptides, and the like.
  • the peptides or proteins contain the desired number of amino acid residues having free amino groups in their side chains; however, other functional groups, such as sulfhydryl groups or hydroxyl groups can also be used to obtain stable linkages.
  • the carbohydrate ligands of the invention may be oxidized to contain carboxyl groups at the reducing terminus which can then be derivatized with either free amino groups to form amides or with hydroxyl groups to form esters.
  • the compounds of the invention of Formula 1 may be synthesized using an intermediate of Formula 2.
  • the intermediate of Formula 2 in one embodiment, can be prepared directly from D-lactose using standard procedures. In this conversion, D-lactose is converted to the octaacetate in crystalline form, in over 95% yield in the method described by Hudson, C, and Kuns, A., J Am Chem Soc (1925) 47:2052.
  • the octaacetate is, in turn, converted in more than 90% yield by the method of Hudson, C. (supra) or of Fischer, E. and Fischer, H., Ber (1910) 43:2521 to the corresponding lactosyl bromide, also a crystalline compound.
  • the protected lactosyl bromide is converted by the method of Jansson, K., et al., J Org Chem (1988) 53:5629, in over 60% yield to the corresponding acylated trimethylsilyl ethyl lactose, which can be deprotected by deacylation in quantitative yield to obtain 2-(trimethylsilyl)ethyl lactoside, 2-(trimethylsilyl)ethyl ⁇ -D- galactopyranosyl- ⁇ -D glucopyranoside.
  • Alternative protecting groups at position 1 of the disaccharide may also be used.
  • R 7 is a protecting group, preferably SE or Bn, wherein SE represents -CH 2 CH 2 SiMe 3 and Bn is benzyl.
  • Reaction Scheme 1 outlines the formation of one embodiment of the compounds of Formula 2 from this intermediate, where Bz represents benzoyl: Reaction Scheme 1
  • step 1 of the reaction scheme the protected lactose, e.g., the trimethylsilyl ethyl derivative, is treated with an excess of 2,2-dimethoxypropane and dry camphor sulfonic acid is added to the reaction mixture which is stirred for 2-3 days at about room temperature.
  • a suitable base such as triethylamine is added and stirring continued for 10-20 minutes; the mixture is then concentrated to dryness and the base removed.
  • the method employed is that of D. Beith-Halahmi et al., Carbohvdr.
  • the intermediate 1_ m &y then be further derivatized at the free hydroxyl at the 3-position of the glucoside residue or this position may be protected and the compound deprotected at positions 3 and 4 of the galactosyl residue and further derivatized at position 3. Position 4 of the galactosyl residue is relatively unreactive.
  • Reaction Scheme 2A A typical scheme for utilization of this key intermediate 1_ is shown in Reaction Scheme 2A. (In this scheme, Bz is benzoyl (PhCO-) and Bn is benzyl (PhCH 2 -).
  • the intermediate 1_ is converted in two steps to intermediate JO by treatment under suitable conditions with protected methyl 1- thio-L-fucoside.
  • the reaction is conducted in a nonaqueous aproctic solvent in the presence of cupric bromide, tetrabutylammonium bromide and molecular sieve.
  • cupric bromide, tetrabutylammonium bromide and molecular sieve S. Sato, et al., Carbohvdr. Res. (1986) 155.C6
  • the resultant compound shown as .H) is then selectively acetylated at position 4 of D-galactopyranosyl residue by the way of its 3,4- orthoester, according to literature procedure, without isolation of the intermediate (R.U. Lemieux and H.
  • intermediate 11. may be phosphorylated to yield intermediate .14 which upon deacylation and hydrogenation yields the final product J ⁇ .
  • This compound would be expected to act as a selectin ligand.
  • alkyl refers to a saturated straight or branched chain or cyclic hydrocarbyl residue containing 1-6C; lower alkyl is similarly defined but containing only 1-4C.
  • alkylaryl is of the formula (CH 2 ) m -Ar wherein m is 1-10 and Ar is a mono- or bicyclic aromatic residue optionally containing one or more heteroatoms.
  • Ar include phenyl, naphthyl, quinolyl, pyridyl, pyrimidinyl, benzthiazoyl, benzimidazoyl, and the like.
  • R7 is a protecting group suitable for saccharide residues.
  • Typical protecting groups include benzyl, benzoyl, various silylalkyl groups, such as trimethylsilylethyl (SE), and the like.
  • Exemplary compounds of formula 1 of the invention are those wherein R 3 is S0 4 -2, P0 4 -2, or other similar charged moiety.
  • Additional exemplary compounds of formula 1 include those wherein X is: 6-methyl-3,4,5-trihydroxypyran-2-yl,
  • R 1 are H or methyl
  • Y is H, OH, OCH 3 or OAc
  • X is -CH 2 (CHOH) 3 H, 3,4,5-trihydroxypyran-2-yl, 3,4,5-trihydroxy-6-methylpyran-2-yl, 3,4,5- trimethoxypyran-2-yl, 3,4,5-trimethoxy-6-methylpyran-2-yl, 3,4,5-trihydroxyfuran-2- yl, 3,4,5-trimethoxyfuran-2-yl, 2,3,4-trihydroxybenzoyl, or 2,3,4- trihydroxynaphthoyl; and R 3 is S0 4 -2, P0 4 -2, or other similar charged moiety.
  • R 1 are H
  • R 2 is H
  • Y is H
  • OR 1 or lower alkyl
  • those compounds of formula 2 which represent intermediates preferred forms are those wherein the protecting groups represented by R 6 are benzyl or benzoyl, the protecting group represented by R 7 is trimethylsilylethyl or benzyl, and wherein Y 1 is H, OR 6 wherein R 6 is benzyl or benzoyl as set forth above, and where the free hydroxyl group(s) is at position 3 of the glucosyl moiety or positions 3 and 4 of the galactosyl moiety.
  • An additional preferred protecting group for positions 3 and 4 of the galactosyl moiety is isopropylidene.
  • T.l.c. (8.5:1.5 toluene-ethyl acetate) revealed the presence of a major product, faster-migrating than the starting acetal. A small proportion of a still faster-migrating product (pentabenzoate) was also revealed by t.l.c.
  • the mixture was poured into ice-water and extracted with dichloromethane. The dichloromethane solution was successively washed with water, aqueous NaHCO 3 , and water, dried (NajSO , and concentrated.
  • Cupric bromide (2.6g, 11.7 mmol), and tetrabutylammoni ⁇ m bromide (3.77g, 11.7 mmol) were added , and the stirring was continued for a total of 48h at room temperature, additional amounts of 8 (2.34g, 5.09 mmol, in 60 mL of 5:1 dichloroethane-N,N-dimethylformamide), cupric bromide (1.3g, 5.85 mmol), tetrabutylammonium bromide (1.9g, 5.85 mmol) and 4A molecular sieves (3g) being added after 24h. T.l.c.
  • the mixture was filtered ( Celite bed) directly onto Amberlite IR 120 (Na + ) cation-exchange resin, and the solids thoroughly washed with aqueous methanol. After stirring with the resin for lh, the mixture was filtered and concentrated to a small volume, which was applied to a column of silica gel and eluted with 5:4:1 chloroform-methanol- water. Fractions corresponding to the product were pooled, concentrated to a small volume and treated with Amberlite IR 120 (Na + ) cation-exchange resin.
  • Example 13 Preparation of a Multivalent Ligand, N,6N,6N' Tris (20) Lys-Tyr-Lys Compound 13a or 13b, prepared in Example 12, may be derivatized to the peptide Lys-Tyr-Lys to obtain the trivalent conjugate derivatized at the two ⁇ - amino lysine groups and the ⁇ -amino N-terminal of the peptide.
  • the results would show the formation of the derivatized peptide as containing 1, 2 or 3 moieties of compound 13a or 13b.
  • the trivalent derivative would be especially effective in inhibiting the binding of lactose to hepatocytes in an assay conducted as described by Lee, R. et al., Biochem (1984) 23:4255.
  • Lactose Derivatives Compounds 13a and 13b were tested for their capacity to bind to E and L selectin.
  • the ELISA assay used consists of evaporating 2,3 sLex glycolipid, at 25 picomoles per well, onto microtiter wells, and then washing the excess off with water. The wells are blocked with 5% BSA at room temperature for an hour and then washed with PBS containing imM Ca.
  • 13a and 13b were added at final concentrations ranging from 1.5 to 5.0 mM to the soluble receptor and allowed to react at 37 °C for 45 minutes.
  • the solutions were then placed in the microtiter wells that had been washed after being blocked, and the plates incubated at 37 C for 45 minutes to allow the soluble receptor to bind to the known natural ligand, 2,3 sLex glycolipid.
  • the positive control was the signal produced by soluble "multivalent" receptor reacted with only the ligand evaporated to the microtiter well. This was considered "100 % binding.”
  • the signal produced by receptor previously reacted with inhibitor is divided by the signal produced by the positive control, multiplied by 100, to calculate % receptor bound in the presence of the inhibitor. The reciprocal of this is % inhibition.

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Abstract

L'invention se rapporte à des composés et à des procédés de préparation de ces composés, lesquels répondent à la formule (1) et se lient à des récepteurs de sélectine, modulant ainsi la progression des inflammations, du cancer et de maladies apparentées en modulant les phénomènes d'adhésion cellule-cellule, formule dans laquelle chaque R représente indépendamment H ou alkyle inférieur (1-4C); R2 représente H, OH, ou alkyle inférieur (1-4C), alkylaryle ou un ou plusieurs restes de saccharide supplémentaires; R3 représente une fraction charge négative comprenant SO4--, PO4--, ou un groupe apparenté; Y représente H ou alkyle inférieur (1-4C); et X représente -CHR?4(CHOR1)¿2CHR?5OR1, R4 et R5¿ représentent chacun indépendamment H, alkyle inférieur (1-4C) ou constituent ensemble un noyau à cinq ou six éléments contenant éventuellement un hétéroatome choisi dans le groupe composé de O, S et NR1; ledit noyau à cinq ou six éléments étant éventuellement substitué par un substituant choisi dans le groupe composé de R1, CH2OR?1, OR1, OOCR1, NR1¿2, NHCOR?1 et SR1¿, à condition que si X représente un substituant hexose, R3 et R4 ne peuvent pas ensemble constituer un substituant hexose.
EP93916790A 1992-06-29 1993-06-25 Derives de lactose substitues utilises comme inhibiteurs d'adhesion cellulaire. Withdrawn EP0648223A4 (fr)

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US91070992A 1992-06-29 1992-06-29
US910709 1992-06-29
PCT/US1993/006110 WO1994000477A1 (fr) 1992-06-29 1993-06-25 Derives de lactose substitues utilises comme inhibiteurs d'adhesion cellulaire

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Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5591835A (en) * 1992-06-29 1997-01-07 Glycomed Incorporated Substituted lactose derivatives
US5620864A (en) * 1992-06-29 1997-04-15 Health Research, Inc. Acceptor for fucosyl transferase
US5783693A (en) * 1993-11-19 1998-07-21 The Regents Of The University Of California Methods for synthesizing sulfated disaccharide inhibitors of selectins
US5444050A (en) * 1994-04-29 1995-08-22 Texas Biotechnology Corporation Binding of E-selectin or P-selectin to sialyl Lewisx or sialyl-Lewisa
JPH0899989A (ja) * 1994-09-30 1996-04-16 Akira Hasegawa 新規糖脂質誘導体およびその製造用中間体
DE4436164A1 (de) * 1994-10-10 1996-04-11 Hoechst Ag Neue Kohlenhydratkonjugate als Inhibitoren der Zelladhäsion
US5962424A (en) * 1995-02-21 1999-10-05 Arch Development Corporation Methods and compositions for targeting selectins
WO1997000881A1 (fr) * 1995-06-22 1997-01-09 Nippon Shinyaku Co., Ltd. Derives de la moranoline
CA2227013A1 (fr) 1995-07-14 1997-02-06 Glycotech Corp. Composes et methodes de traitement de cancers lies au recepteur egf et purification du recepteur egf
JPH0952902A (ja) * 1995-08-09 1997-02-25 Daikin Ind Ltd フッ素含有シアリルルイスｘ誘導体及びその合成中間体
EP1210125A2 (fr) * 1999-09-08 2002-06-05 INSTITUT FÜR DIAGNOSTIKFORSCHUNG GmbH AN DER FREIEN UNIVERSITÄT BERLIN Agents de contraste de type l-selectine
FR2929510B1 (fr) * 2008-04-02 2011-01-21 Lvmh Rech Utilisation d'au moins un glycoside d'alkyle en tant qu'agen qu'agent anti-vieillissement et/ou calmant des peaux sensibles dans des compositions cosmetiques, et methodes de soin cosmetique utilisant les dites compositions.

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010796A1 (fr) * 1991-11-27 1993-06-10 Glycomed, Inc. Derives de lactose et de lactosamine a substitution utilises comme inhibiteurs d'adhesion cellulaire
EP0579196A1 (fr) * 1992-07-15 1994-01-19 The Nisshin Oil Mills, Ltd. Dérivés de glycolipides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010796A1 (fr) * 1991-11-27 1993-06-10 Glycomed, Inc. Derives de lactose et de lactosamine a substitution utilises comme inhibiteurs d'adhesion cellulaire
EP0579196A1 (fr) * 1992-07-15 1994-01-19 The Nisshin Oil Mills, Ltd. Dérivés de glycolipides

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, vol. 31, 29 September 1992, pages 9126-9131, XP000566479 CHUN-TING YUEN ET AL: "Novel Sulfated Ligands for the Cell Adhesion Molecule E-Selectin..." *
CARBOHYDRATE RESEARCH, vol. 155, 1986, pages c6-c10, XP002002609 S. SATO ET AL: "An efficient approach to O-glycosides..." *
CARBOHYDRATE RESEARCH, vol. 174, 1988, pages 23-36, XP002002611 J. BREG ET AL: "Characterisation of Four Lactose Monophosphates..." *
CELL, vol. 67, 4 October 1991, pages 35-44, XP002002610 A. ARUFFO ET AL: "CD62/P-Selectin Recognition of Myeloid and Tumor Cell Sulfatides" *
J. BIOL. CHEM., vol. 267, 25 November 1992, pages 23806-23814, XP000566486 E.V. CHANDRASEKARAN ET AL: "Ovarian Cancer alpha 1,3-L-Fucosyltransferase" *
JOURNAL OF THE CHEMICAL SOCIETY, CHEMICAL COMMUNICATIONS, no. 13, 1 July 1991, pages 870-872, XP000562674 NICOLAOU K C ET AL: "STEREOCONTROLLED SYNTHESIS OF SIALYL LEX, THE OLIGOSACCHARIDE BINDING LIGAND TO ELAM-1 (SIALYL = N-ACETYLNEURAMIN)" *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 88, no. 22, 15 November 1991, pages 10372-10376, XP000562667 TYRRELL D ET AL: "STRUCTURAL REQUIREMENTS FOR THE CARBOHYDRATE LIGAND OF E-SELECTIN" *
See also references of WO9400477A1 *

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WO1994000477A1 (fr) 1994-01-06
EP0648223A4 (fr) 1996-07-31
CA2138645A1 (fr) 1994-01-06
AU4652693A (en) 1994-01-24
AU678373B2 (en) 1997-05-29
JPH08500820A (ja) 1996-01-30

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