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WO1997031007A1 - MIMETIQUES DE SIALYL LEWISx CONTENANT DES SQUELETTES DE FLAVANOIDE - Google Patents

MIMETIQUES DE SIALYL LEWISx CONTENANT DES SQUELETTES DE FLAVANOIDE Download PDF

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Publication number
WO1997031007A1
WO1997031007A1 PCT/US1997/002951 US9702951W WO9731007A1 WO 1997031007 A1 WO1997031007 A1 WO 1997031007A1 US 9702951 W US9702951 W US 9702951W WO 9731007 A1 WO9731007 A1 WO 9731007A1
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Prior art keywords
group
compounds
direct link
independently selected
reaction
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PCT/US1997/002951
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English (en)
Inventor
Mark B. Anderson
Daniel E. Levy
Peng Cho Tang
John H. Musser
Narasinga Rao
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Glycomed Incorporated
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Application filed by Glycomed Incorporated filed Critical Glycomed Incorporated
Priority to AU19752/97A priority Critical patent/AU1975297A/en
Priority to EP97907858A priority patent/EP0888369A1/fr
Publication of WO1997031007A1 publication Critical patent/WO1997031007A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans
    • C07H17/07—Benzo[b]pyran-4-ones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/32—2,3-Dihydro derivatives, e.g. flavanones

Definitions

  • This invention relates generally to the field of medicinal chemistry, and specifically to medicaments that are characterized by their capacity to bind to one or more of the three known selectins: E, , and P-selectins.
  • the medicaments consist of substituted pseudo-oligosaccharides wherein three chemical moieties are covendingly linked in the following order : sialic acid, or an analogue, deriva ⁇ tive, or mimic thereof, a flavanoid spacer, and fucose or an analogue, derivative, or mimic thereof.
  • Such medica ⁇ ments have significant applications for diagnosis or prophylactic or therapeutic treatment of certain diseases including cancer, autoimmunity, and inflammation.
  • L-Selectin L-Selectin
  • E-Selectin ⁇ LECAM-2 EIAM-1
  • P-Selectin LCAM-3, G P-140, PADGEM
  • P-selectm has been shown to be centrally involved particularly as related to acute lung injury. Mulligan et al . have reported strong protective effects using anti-P-selectin antibody in a rodent lung injury model. (Mulligan, M. S. et al . , J. Clin. Invest., (1991) 9__:1600, Mulligan, M. S. et al . , Nature (1993) 364:149) . A central role of P- selectin in inflammation and thrombosis has been demon ⁇ strated by Palabrica et al . (Palabrica, T. et al . , Nature (1992) 3_59:843) .
  • E-selectm is particularly interesting because of its transient expression on endo ⁇ thelial cells in response to IL-1 or TNF (Bevilacqua et al . , Science (1989) 243 : 1160) ⁇
  • the time course of this induced expression(2-8 h) suggests a role for this recep ⁇ tor m initial neutrophil extravasation m response to infection and injury.
  • Gundel et al . have shown that antibody to E-selectm blocks the influx of neutro ⁇ phils in a primate model of asthma and thus is beneficial for preventing airway obstruction resulting from the inflammatory response. (Gundel R. H. et al . , J. Clin. Invest . (1991) 88:1407) .
  • the PCT publication claims methods of identifying E- selectin ligands, as well as methods of inhibiting adhesion between leukocytes and endothelial cells using such ligands and specifically refers to MILAs which are described as molecules involved in leukocyte adhesion to endothelial cells.
  • selectin ligands describe the use of L-selectin as an indicator of neutrophil activation (Butcher et al., U.S. Patent 5,316,- 913 issued May 31, 1994), and assays for inhibition of leukocyte adhesion (Rosen et al . , U.S. Patent 5,318,890 issued June 7, 1994) .
  • the ligand for E-selectin, sLe x consists of at least sialic acid, fucose, and N-acetyl lactosamine.
  • Lactosamine consists of galactose and 2- amino-2-deoxyglucose.
  • Sialic acid and fucose are bound to the galactose and glucosamine moieties of lactosamine, respectively.
  • Ligands that bind to the other selectins share similar structural features.
  • the selectin family of adhesion molecules participate in acute inflammation by initiating neutrophil rolling on activated endothelial cells. This is particularly evident in studies of ischemia reperfusion injury, where P-select- in appears to be important in neutrophil recruitment to damaged tissue.
  • the presence of L-selectin and E- or P- selectin ligands on mononuclear cells has implicated these receptor-ligand interactions in chronic inflammation. This has been supported by the finding of chronic expres ⁇ sion of E-selectin in dermatologic conditions, and P- selectin expression on joint synovial endothelium derived from rheumatoid arthritis patients. L. Lasky Annu. Rev. Biochem.
  • a first object of the invention is the description of medicaments that are selectin ligand mimetics that bind to certain selectins wherein the mimetics lack the lactose core structure of the natural selectin ligand, sialyl Lewis" (sLe ) , and have substituted in its place a flava ⁇ noid moiety, relative to sLe .
  • Such invention compounds are represented by the following general structural formulae I and II :
  • R 2 is selected from the group consisting of -H and
  • each R 3 is independently selected from the group consisting of -H, lower alkyl, saturated or unsaturated carboxylic acids of 1 to 4 carbon atoms, optionally substituted with 1 to 2 hydroxyl groups, and esters and amides thereof, with the proviso that both of R 3 do not contain a carboxylic acid;
  • R 2' , R 3' , R 4' , R 5' , R 6' , R 5 , R 6 , R 7 , and R 8 are independently selected from the group consisting of
  • Y-B is selected from the group consisting of
  • W is selected from the group consisting of a direct link, -0-, -N, -S-, -NH- , and -NAc- ;
  • U is selected from the group consisting of -R 9 , -CH 2 OR 10 , -CH 2 0-protecting group, -COOR 11 , -CON(R n ) 2 , and -COOM;
  • R 9 is lower alkyl; each n is independently selected from the group 0, 1, 2 , and 3 ; each m is independently selected from the group 0, 1, 2 , 3, and 4 ; each q is independently selected from the group 0, 1, and 2 ; each s is independently selected from the group 1 , 2 , and 3 ; each z is independently selected from the group 1 and 2; each t is independently selected from the group 1 and 2, with the proviso that when is -N , then t is 2, and at all other definitions of W, t is 1;
  • R u is independently selected from the group consisting of -H, lower alkyl, cyclic alkyl of 5 to 6 carbon atoms, heterocyclic alkyl of 4 to 5 carbon atoms and 1 to 2 heteroatoms, lower aryl and lower aralkyl;
  • R 12 is selected from the group consisting of -N(R ) 2 , and -SR 11 ;
  • R 13 is selected from the group consiting of R 11 , and M;
  • R 14 is selected from the group consisting of -H, and - OR 10 , with the proviso that when z is 2, then together the two R 14 groups may form a double bond;
  • R 15 is independently selected from the group consisting of -R 11 and -COOH.
  • M is selected from the group consisting of Na * , K * , Mg 2 ⁇ and Ca 2 ⁇ -
  • M' is selected from the group consisting of -H, -M, and R 9 ;
  • X is selected from the group consisting of -0-, -S-, -C(R 11 ) 2 -, and -N(R n )-; and pharmaceutically acceptable salts thereof with the provisos that:
  • R 2 , R 3 , R 4 , R 5' , and R 6 may be a direct link to B; (e) at most three of R 2 R 3 , R 4 , R c , and R 6 may be independently selected from the group consisting of -OH and ether moieties; (f) no more than three of R 2' R 3' , R 4' , R 5' , and R 6' may contain a B group;
  • R 2' p R 3' , R 4' , R 5' , and R 6' are -H;
  • no more than two of R 5 ,R 6 , R 7 , and R 8 may be Y-B when W is a direct link;
  • R 6 and R 8 may be a direct link to B, and R 6 or R 8 is a direct link to B, then R 7 must be -OH.
  • R 5 , R 6 R 7 ,and R 8 may be independently selected from the group consisting of -OH and ether moieties;
  • R 5 when none of R 2' , R 3' , R 4 ' , R 5' , R 6' , R 2 , R 3 , R 6 , R 7 , and R 8 is a substituent containing an A group, then R 5 must be -OH; (o) when R 5 is not -OH, then at least one of R 5 , R 6 , R 7 , and R ⁇ is a substituent containing an A group, or at least one R 3 is a substituent containing a carboxylic acid, ester, or amide thereof, or at least one of R 2' , R 3' R 4' , R s' , and R 6' contains an A group where A is not B; (p) at least one of R 2' R 3' , R 4' , R 5' , R 6' , R s , R c , R 7 , and R 8 is a substituent containing a B group, and at least one is a substituent containing an A group where
  • a second object of the invention is a description of certain novel medicaments that incorporate newly discovered physical/chemical properties associated with sLe x , such that the medicaments have a three-dimensionally stable configuration for the presentation of the functional groups of sLe x , sialic acid and fucose, that facilitates binding between those groups and the selectins.
  • a third object of the invention is to provide a composition comprising selectin ligand medicaments bound to a detectable label and/or bound to a pharmaceutically active drug such as an anti-inflammatory drug.
  • a fourth object of the invention is to provide a pharmaceutical formulation containing selectin ligand medicaments which is useful in treating certain diseases.
  • a fifth object of the invention is to provide a description of methods to treat or diagnose disease.
  • a sixth object of the invention is to provide compositions and methods to determine the site of inflammation by administering labeled formulations of the type referred to above.
  • Another object of the invention is that the ligands can be labeled and the labeled ligands used in an assay to detect the presence of selectins in a sample.
  • Sialic acids refer to the family of amino sugars containing 9 or more carbon atoms, N- and 0- substituted derivatives of neuraminic acid.
  • Kemp's acid refers to 1, 3 , 5-trimethyl-l, 3, 5- cyclohexane-tricarboxylic acid, where each acid is axial.
  • N-acetyl neuraminic acid refers to 5- (acetylamino) -3, 5- dideoxy-D-glycero-D-galacto-2-nonulosonic acid:
  • ex form refers to standard nomenclature representing the configuration of the anomeric position of an O- or C- glycoside.
  • ⁇ form refers to standard nomenclature representing the configuration of the anomeric position of an O- or C-glycoside.
  • Amino refers to -NR 2 where each R is independently selected from -H, lower alkyl, lower aryl, and lower aralkyl .
  • Alkyl refers to saturated hydrocarbons, which may be straight chain, branched, cyclic, or alicyclic. Preferably the alkyl group contains 1 to 10 carbon atoms. Most preferred is 1 to 4 carbon atoms.
  • Lower alkyl refers to branched or straight chain alkyl of 1 to 4 carbon atoms.
  • Alkoxy refers to -OR, where R is an alkyl group.
  • Lower alkoxy refers to -OR where R is lower alkyl .
  • Aryl refers to aromatic groups which have one ring having a conjugated pi electron system and includes carbocyclic aryl, and heterocyclic aryl, both of which may be optionally substituted.
  • Lower aryl refers to an aryl containing up to 6 carbon atoms, and may be optionally substituted.
  • Carbocyclic aryl groups are groups wherein the ring atoms are carbon atoms .
  • Heterocyclic aryl groups are groups having from 1 to 2 heteroatoms in the ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include nitrogen, oxygen, and sulfur. Suitable heterocyclic aryl groups include pyridyl, furanyl, thienyl, pyrrolyl, and the like all optionally substituted. Heteroaryl is the same as heterocyclic aryl.
  • Alicyclic refers to groups which combine the properties of aliphatic and cyclic alkyl groups. For examp
  • aryl groups refers to either no substitution or substitution by one to three substituents independently selected from lower alkyl, halo, carboxylic acids, esters, -N0 2 , and lower perhaloalkyl .
  • Alkyl refers to an alkyl group substituted with an aryl group, which may be optionally substituted. Benzyl is a suitable aralkyl group. Lower aralkyl refers to up to and including 8 carbon atoms, and may be optionally substituted. The aralkyl group is attached through the alkyl portion of the group.
  • Alkenyl refers to unsaturated groups which contain at least one carbon-carbon double bond and includes straight-chain, branched chain, and cyclic groups. The double bond may be exo to the chain.
  • Alkoxyaryl refers to aryl substituted with alkoxy group.
  • Alkynyl refers to unsaturated groups which contain at least one carbon triple bond and includes straigh - chain, branched chain, and cyclic groups.
  • Aryloxy refers to -O-aryl.
  • Alkoxy refers to -O-aralkyl .
  • Carboxylic acid refers to -COOH.
  • Ester refers to -COOR where R is lower alkyl, lower aryl, and lower aralkyl;
  • Amide refers to -CONR 2 where each R is independently selected from hydrogen, lower alkyl, lower aryl, and lower aralkyl. Preferably at least one R is hydrogen.
  • M refers to a cationic metal selected from Na*, K ⁇ Mg 2* , and Ca t . Where M is associated with -COO " , it is preferably a plus one cation, and more preferably Na*.
  • Protecting group refers to a group protecting one or several inherent functional groups . Suitable “protecting groups” will depend on the functionality and particular chemistry used to construct the library. Examples of suitable functional protecting groups will be readily apparent to skilled artisans, and are described, for example, in Greene and Wutz, Protecting Groups in Organic Synthesis, 2d ed., John Wiley & Sons, NY (1991) , which is incorporated herein by reference. Suitable -0- protecting groups can be found in the above book. Preferred such protecting groups include acetate and benzyl.
  • Carbohydrate refers to a chemical moiety comprising the general composition (C) n (H 2 0) n , including, but not limited to glucose, galactose, fucose, fructose, sac ⁇ charose, mannose, arabinose, xylose, sorbose, lactose, and derivatives thereof, including but not limited to com ⁇ pounds which have other elemental compositions, such as aldonic acids, uronic acids, desoxysugar ⁇ , or which contain additional elements or moieties, such as amino sugars wherein n is typically 4, 5, 6, 7 atoms and wherein the oxygen atom in the carbohydrate can be replaced by a heteroatom such as nitrogen, sulfur, carbon etc.
  • a carbohydrate as used herein is understood to include chemical structures wherein "H" of any hydroxy group is replaced by any chemically compatible moiety "R" , which can be any monomer, oligomer or polymer in the meaning as used herein.
  • Carbohydrates can be saturated or unsat- urated. Carbohydrates may be charged or uncharged. Suitable charged carbohydrates include galacturonic acid, glucuronic acid, and sialic acid.
  • “Carbohydrate unit” is a monomer comprising a mono- saccharide.
  • Carbon glycoside is a carbohydrate derivative wherein the anomeric position does not have an oxygen but a carbon substituent.
  • Heteroatom glycoside is a carbohydrate wherein the oxygen at the anomeric position is replaced by an atom other than oxygen, including carbon, nitrogen, sulfur, phosphorous and silicon.
  • Identity tag is any detectable attribute that provides a means to elucidate the structure of an individual oligomer in a labeled synthetic oligomer library. For example, an identifier tag can be used to identify the resulting products in the synthesis of a labeled synthetic oligomer library.
  • Named Reactions are chemical reactions which are chemical standard reactions known by those of ordinary skill in the art, including but not limited to the Alper Reaction, Barbier Reaction, Claisen-Ireland Reaction, Cope Rearrangement, Delepine Amine synthesis, Gewald
  • Cope Reaction or “Cope” refers to the 3, 3 sigmatropic rearrangement and includes the Claisen-Ireland Reaction and all forms of the Cope rearrangement.
  • Oletaccharide or “polysaccharide” refers to carbohydrates, including carbon glycosides, comprising a plurality of monosaccharides.
  • Synthetic chemical library is a collection of random and semi-random synthetic molecules wherein each member of such library is produced by chemical or enzymatic synthesis.
  • a “Synthesis support” is a material having a rigid or semi-rigid surface and having functional groups or linkers.
  • a synthesis support may be capable of being derivatized with functional groups or linkers that are suitable for carrying out synthesis reactions.
  • Such materials will preferably take the form of small beads, pellets, disks, capillaries, hollow fibers, needles, solid fibers, cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with polyethylene glycol divinylbenzene, grafted co-poly beads, poly-acrylamide beads, latex beads, dimethylacryl- amide beads optionally cross-linked with N,N' -bis-acryloyl ethylene diamine, glass particles coated with a hydro- phobic polymer, or other convenient forms.
  • Transformation event is any event that results in a change of chemical structure of a compound, monomer, an oligomer or polymer.
  • a "transforma- tion event” or “reaction” may be mediated by physical, chemical, enzymatic, biological or other means, or a combination of means, including but not limited to, photo, chemical, enzymatic or biologically mediated isomerization or cleavage, photo, chemical, enzymatic or biologically mediated side group or functional group addition, removal or modification, changes in temperature, changes in pressure, and the like.
  • transformation event includes, but is not limited to, events that result in an increase in molecular weight of a monomer, an oligomer or polymer, such as, for example, addition of one or a plurality of monomers, addition of solvent or gas, or coordination of metal or other inorganic substrates such as, for example, zeolities.
  • a “transformation event” or “reaction” may also result in a decrease in molecular weight of an oligomer or polymer, such as, for example, de-hydrogenation of an alcohol to from an alkene or enzymatic hydrolysis of an ester or amide.
  • Transforma- tion events also include events that result in no net change in molecular weight of a monomer, an oligomer or polymer, such as, for example, stereochemistry changes at one or a plurality of a chiral centers, Claissen rearrangement, Ireland rearrangement, or Cope rearrangement and other events as will become apparent to those skilled in the art upon review of this disclosure.
  • Heterocyclic alkyl refers to a cyclic alkyl group in which one to three of the ring atoms are a heteroatom and the remaining ring atoms are carbon atoms. Suitable heteroatoms are nitrogen, oxygen, and sulfur. Suitable heterocyclic alkyl groups are morpholine, piperadine, and piperazine.
  • “Flavanoid” refers to bicyclic structures as depicted in Formula I and II and include flavones, ⁇ soflavones, flavones, isoflavones, chromones, and chromanones.
  • the dotted line in structure I and II is an optional double bond.
  • the bicyclic structure has an aromatic carbocyclic ring fused to a heterocyclic ring.
  • Adjacent position refers to the next carbon atom on the phenyl or flavanoid ring.
  • R 3' and R 1' are adjacent to R 2' , in the phenyl structure depicted below:
  • R 2 is adjacent to R 3
  • R 6 is adjacent to R 5 and R 7 .
  • Phenyl structure refers to the aromatic group above.
  • “Ether moiety” refers to any substituent or group linked through an oxygen.
  • -Ar- refers to a phenyl, optionally substituted.
  • -alk- refers to an alkyl linking group which is selected from lower alkyl, and cycloalkyl .
  • Suitable "-alk-” groups include -C(CH 3 ) 2 -, and — ⁇ >—
  • Halo refers to halogen atoms -F, -Cl, -Br, and -I.
  • “Cycloalkyl” refers to cyclic alkyl groups and include cyclopropyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • pharmaceutically acceptable salt includes salts of compounds of formulae I and II derived from the combination of a compound of this invention and an organic or inorganic acid or base. The compounds of formula I are useful in both the free acid and salt form.
  • Figure 1 is a cross-sectional schematic view showing the interaction between white blood cells and activated endothelial cells.
  • Figure 2 is a cross-sectional schematic view showing how compounds of the invention would be used as pharmaceu- ticals to block selectin ligand interactions.
  • BSA bovine serum albumin
  • DEAE diethylaminoethyl
  • DMSO dimethylsulfoxide
  • DMF N,N-dimethylforamide
  • DCE dichloroethane
  • E-selectin or ELAM-1 endothelial/leukocyte adhesion molecule-1
  • HPTLC high performance thin layer chromatography
  • L-selectin or LECAM-1 leukocyte/endothelial cell adhesion molecule-1
  • MOPS 3- (N-Morpholino) -propanesulfonic acid
  • NANA N- acetylneuraminic acid
  • PVC polyvinylchloride,* TLC, thin layer chromatography
  • TFA trifluoro-acetic acid
  • Tris tris (hydroxy-methyl) aminomethane.
  • E-selectin has a lectin like domain that recognizes the Sialyl Lewis x (sLe ) tetrasaccharide epitope as shown below in Structure III.
  • a key step in developing the compounds of the present invention was the realization that both sLe x and sLe a share a structural similarity in their three dimensional arrangements.
  • sialic acid and fucose two functional epitopes in these tetrasaccharides, are juxtaposed in space in a way suitable for recognition by the selectins.
  • sialic acid and fucose two functional epitopes in these tetrasaccharides
  • fucose two functional epitopes in these tetrasaccharides
  • the number of atoms refers to the number of atoms between the 0- glycoside of sialic acid and the O-glycoside of fucose. For instance, a close structural examination of sLe x
  • Fucose are linked through six atoms (Nos. 1-6) or eight atoms (Nos. l-vin) as shown in Structure III (a) below
  • R is -NHAc or -OH.
  • the compounds of the present invention possess an acid functionality mimic which is preferably 8-14A, and more preferably 9-11A from a fucose or fucose mimic. This distance is measured from the carbonyl carbon of the acid mimic to the C-3 carbon of fucose or its equivalent on its mimic .
  • replacement of the lactose core with a partially or completely rigid core, while still juxtaposing the two functional epitopes in space in a way suitable for recognition by the selectins would lead to compounds, such as those described and claimed herein, that maintain their selectin binding activity.
  • the Flavanoid family of compounds offer considerable diversity and facility in terms of attachment of suitable groups to satisfy the spacial requirements for selectin ligand binding.
  • the phenyl at R 2 could possess the A and/or B groups in an appropriate position for recognition by selectins.
  • mimics of sialic acid include moieties containing carboxylic acids, esters and amides. It also includes a vmylagous acid which can mimic the acid functionality, such the group shown below:
  • sialic acid mimics also referred to as "A" in the general description, can be in the form of moieties containing sulfates, sulfonates, phosphates, phosphonates, sulfonamides, nitrates, other carboxylic acid equivalents, and the like.
  • Other acid mimics include B groups, particularly B groups which contain acids and sulfates.
  • the compounds of the present invention are designed to provide a three-dimensionally stable configuration for functional groups on sialic acid and fucose moieties or their analogues or mimics so as to allow for binding between those functional groups and receptors on natural selectins.
  • the compounds are represented by the following general structural formulae I and II :
  • R 2 is selected consisting of -H and
  • each R 3 is independently selected from the group consisting of -H, lower alkyl, saturated or unsaturated carboxylic acids of 1 to 4 carbon atoms, optionally substituted with 1 to 2 hydroxyl groups, and esters and amides thereof, with the proviso that both of R 3 do not contain a carboxylic acid;
  • R 4 R 5 R 6 R 5 , R 6 , R 7 , and R ⁇ are independently selected from the group consisting of
  • W is selected from the group consisting of a direct link, -0-, -N, -S-, -NH-, and -NAc- ;
  • U is selected from the group consisting of -R 9 , -CH 2 OR 10 , -CH 2 0-protect ⁇ ng group, -COOR 11 , -CON(R n ) 2 , and -C00M;
  • R 11 is independently selected from the group consis ⁇ ting of -H, lower alkyl, cyclic alkyl of 5 to 6 carbon atoms, heterocyclic alkyl of 4 to 5 carbon atoms and 1 to 2 heteroatoms, lower aryl and lower aralkyl;
  • R 12 is selected from the group consisting of -N(R 11 ) 2 , and -SR 1 -;
  • R 13 is selected from the group consitmg of R 11 , and M;
  • R 14 is selected from the group consisting of -H, and - OR 10 , with the proviso that when z is 2, then together the two R 14 groups may form a double bond;
  • R 15 is independently selected from the group consisting of -R 11 and -COOH.
  • M is selected from the group consisting of Na + , K*, Mg 2 ⁇ and Ca 2 ⁇ *
  • M' is selected from the group consisting of -H, -M, and R 9 ; and X is selected from the group consisting of -0-,
  • R 2 , R 3 , R 4 , R 5 , and R 6 may be a direct link to A;
  • only one of R 2 , R 3 , R 4 , R 5 , and R 6 may be a direct link to B;
  • R 2 R 3 , R 4 , R 5 , and R* may be independently selected from the group consisting of -OH and ether moieties;
  • no more than three of R 2 R 3 , R 4 , R 5 , and R 6 may contain a B group;
  • R s , R 6 R 7 ,and R B may be independently selected from the group consisting of -OH and ether moieties; (n) when none of R 2' , R 3' , R 4' , R 5' , R 6' , R 2 , R ⁇ R 6 , R 7 , and R 8 is a substituent containing an A group, then R 5 must be -OH;
  • R 7 , and R 8 is a substituent containing an A group, or at least one R 3 is a substituent containing a carboxylic acid, ester, or amide thereof, or at least one of R 2' , R 3' R 4' ,
  • R s' , and R 6' contains an A group where A is not B;
  • R 2' _ R 3 ', R 4' , R 5' , R 6' , R 5 , R 6 , R 7 , and R 8 is a substituent containing a B group, and at least one is a substituent containing an A group where A is not B or R 5 must be -OH;
  • hydro- phobic carriers like ceramide or a ceramide mimic, steroids, diglycerides or phospholipids to form other medically useful molecules.
  • the compounds can act as antagonist ligand molecules, i.e. biochemical blocking agents by binding to selectins and preventing circulating leukocytes from binding to endothelial cells, thereby preventing a primary event involved in certain diseases, including cancer, and particularly metastatic cancers, conditions associated with acute inflammation, such as reperfusion injury, septic shock, hypovolemic or traumatic shock, ARDS, and chronic inflammation diseases such as rheumatoid arthritis and asthma.
  • Agonist ligands have the opposite effect.
  • the compounds of structural formulae I and II can be bound to known drugs, for example anti-inflammatory drugs so as to target the drug-selectin ligand complex to a particular site of disease. Additionally, they can be formulated to provide compositions useful in assaying a sample for the presence of selectins such as E, L and/or P-selectin, or to detect the site of inflammation in a patient, or to treat acute inflammation (or treating the inflammatory symptoms of certain diseases) or other diseases involving the interaction of selectins on appropriate cell types.
  • selectins such as E, L and/or P-selectin
  • R 2' , R 3' , R 4' , R 5 ' , R 6' , R 5 , R 6 , R 7 , and R 8 groups include
  • a and B are shown respectively in formula V and VI .
  • Other examples of A include a or ⁇ or other analogues or derivatives of sialic acid other than the N-acetyl neuraminic acid residue shown in formula V, Kemp's acid, quinic acid, R and S forms of mandelic acid, R and S forms of glyceric acid, R and S forms of lactic acid, propionic and acetic acid, and esters and amides thereof, -S0 3 , -P0 3 .
  • the synthesis of certain analogues of sialic acid is described in U. S. Patent No. 5, 138, 044.
  • V VI Preferred embodiments of the ligands of the invention are those wherein the substituent represented by A or B is an N-acetylneuramyl residue and B is fucose.
  • B Preferred forms of B are the and ⁇ forms of L- fucose as shown m formula VI.
  • the moiety B also includes substituted forms of the following and ⁇ -fucose structure VI :
  • R 17 , R ⁇ and R 19 are each independently -OH, -F, -N(R 9 ) 2 (wherein R 9 is lower alkyl) .
  • moieties for B include modified fucosides such as corresponding carboxylic analogues of fucose; inositol; substituted inositol; imidazole; substituted imidazole; benzimidazole; substituted benzimidazole; Guanidine; pentaerythritol; substituted pentaerythritol; and substituted butanes of the formula -CH 2 -CHR 17 -CHR 18 -CH 2 R 19 wherein R 12 , R 18 , and R 19 are independently -OH, -F or - N(R 9 ) 2 .
  • Preferred B groups include those where s is 1 or 2,
  • R 14 is -H or -OH
  • X is -0-
  • U is -CH 2 OR 10 or -R 9
  • R 10 is - alk-COOH, -S0 3 M, -H, or -alk-COOM.
  • B groups include fucose, galactose, mannose, and arabinose.
  • Preferred s numbers are 1 and 2. Most preferred is 2.
  • Preferred q numbers are 0 and 1.
  • Preferred m numbers are 0 and 1.
  • Preferred n numbers are 0 and 3.
  • Preferred z numbers are 1.
  • Preferred R 10 groups are -H, S0 3 M, -alk-COOR 13 , and -O-carbohydrate. Most preferred are -H, -S0 3 M, and -alk-COOR 13 .
  • Preferred R 11 groups are -H, lower alkyl, and lower aralkyl. Most preferred is -H.
  • a preferred R 12 group is -N(R ) 2 .
  • Preferred R 14 groups are -H and -OH.
  • Preferred R 15 groups are -COOH, -H, and -CH 3 .
  • a preferred M cation is Na * .
  • Preferred M' groups are -H, Na * , and -CH 3 .
  • a preferred X group is -O- .
  • Preferred U groups are -CH 2 OR 10 and -R 9 .
  • Preferred W groups are a direct link and -O- , and t is 1.
  • a preferred R 3 group is saturated or unsaturated carboxylic acid of 1 to 4 carbon atoms, optionally substituted with 1 to 2 hydroxy groups, and esters and amides thereof .
  • a preferred R 2 group is
  • R 3 is a saturated or unsat ⁇ urated carboxylic acid of 1 to 4 carbon atoms, optionally substituted with 1 to 2 hydroxy groups, and esters and amides thereof
  • R 5 is -OH
  • R 14 is -H or -OH
  • X is -0-
  • U is -CH 2 OR 10 or -R 9
  • R 10 is -alk-COOH, -S0 3 M, - H, or -alk-COOM.
  • Particularly preferred are such compounds where s is 2.
  • Also particularly preferred are compounds where R 15 is H.
  • R 3 is saturated or unsaturated carboxylic acid of 1 to 4 carbon atoms, optionally substituted with 1 to 2 hydroxl groups, and esters and amides thereof, R 5 and R 7 are -OH, and R 8 is selected from the group consisting of
  • R 2 is -OH
  • W is a direct link or -O- and t is 1, and where in the B group s is 1 or 2, R 14 is -H or -OH, X is -O- , U is -CH 2 0R 10 or -R 9 , and R 10 is -alk-COOH, S0 3 M, -H, or -alk- COOM.
  • R 15 is -H and s is 2.
  • R 1 is a saturated or unsaturated carboxylic acid of 1 to 4 carbon atoms, optionally substituted with 1 to 2 hydroxyl group, and esters and amides thereof
  • R 2 is -OH
  • W is a direct link or -O- and t is 1
  • B group s is 1 or 2
  • R 14 is -H or -OH
  • X is -O-
  • U is -CH 2 OR 10 or -R 9
  • R 10 is - alk- COOH , - S0 3 M , -H , or - alk - COOM .
  • More pref erred are compounds where R 1 is - CH 2 COOH , and esters and amides thereof.
  • R 15 is -H and s is 2.
  • B group s is 1 or 2
  • R 14 is -H or -OH
  • X is -0-
  • U is -CH 2 OR 10 or -R 9 and R 10 is -alk-COOH, -S0 3 M
  • Preferred compounds include those prepared in the Examples and those found in Table 3.
  • the compounds of formulae I and II can be tested for their ability to bind to a selectin receptor and/or block the binding site of the receptor and thereby prevent a natural ligand from binding to the selectin receptor.
  • a generalized procedure for testing the compounds of formulae I and II is given below.
  • An ELISA assay is preferably used that employs recombinant fusion proteins composed of extracellular portions of the human selectins joined to human lmmuno- globulin heavy chain CH 3 , CH 2 , and hinge regions. See, for example, Walz et al . , Science (1990) 250 : 1132 ; Aruffo et al . , Cell (1991) .67:35; Aruffo et al . , Proc. Natl. Acad. Sci . USA (1992) .89*2292.
  • the assay is well known in the art, and generally consists of the following three steps: I.
  • 2,3 sLe x glycolipid (25 picomole/well) was transferred into microliter wells as solutions and then evaporated off. Excess, which remained unattached, was washed off with water. The wells were then blocked with 5% BSA at room temperature for an hour and then washed with PBS containing 1 mM calcium.
  • invention compounds include those having sialic acid and fucose separated by 4-12 atoms, or sialic acid or analogs, derivatives or mimics of sialic acid separated by 4-12 atoms bound to fucose or analogs, derivatives or mimics thereof.
  • FIG. 1 a cross-sectional view of a blood vessel 1 is shown.
  • the vessel wall 2 is lined internally with endothelial cells 3.
  • the endothelial cells 3 can be activated causing the cells 3 to synthesize E or P-selectin which is displayed in Figure 1 as a triangular surface receptor 4.
  • Both red blood cells 5 and white blood cells (6A, 6B) flow in the vessel 1.
  • the white blood cells 6 display carbohydrate ligands 7 which have chemical and physical characteristics which allow the ligands 7 to bind to the receptors 4. Once the ligand 7 binds to the receptor 4 , the white blood cell 6 is brought through the vessel wall 2 as is shown with the white blood cell 6A.
  • the white blood cells 6B brought into the sur ⁇ rounding tissue 8 can have positive effects, such as fighting infection, and negative effects, such as inflammation.
  • the compounds of formula I are shown as 7A and can adhere to a selectin such as E, L and/or P-selectin by themselves and can be formulated into pharmaceutical compositions, which when administered will effectively block the E, L and/or P- selectin and prevent the adhesion of a ligand 7 connected to a white blood cell 6.
  • a selectin such as E, L and/or P-selectin
  • the selectin family of adhesion molecules participate in acute inflammation by initiating neutrophil rolling on activated endothelial cells.
  • the compounds of formulae I and II may also be labeled using standard radioactive, fluorescent, enzymic or other labels for analytical or diagnostic purposes.
  • a ligand of the invention In order for a ligand of the invention to bind to a selectin receptor such as E, L and/or P-selectm receptor the ligand need not include the identical atoms in the identical configuration as per structural formulae I and II but must have (1) a relatively stable three dimensional conformation as shown in formulae I and II, or (2) a substantially equivalent configuration to that shown in formulae I and II.
  • the equivalency of any other ligand will relate to its physical three dimensional structure and the electron configuration of the molecule, and in particular the charge related characteristics presented by the groups present on the A and B moieties shown m formulae V and VI .
  • Candidate ligands can be assayed for their ability to adhere to E, L or P-selectin.
  • the method comprises attaching candidate ligands of formulae I and II to a substrate surface and then contacting the substrate surface thereon with recombinant cells, that are genetically engineered to express high levels of E, L or P-selectin, for a sufficient time to allow the cells to adhere to the substrate bearing the candidate ligand. Thereafter, centrifugal force or other appropriate methodology is applied so as to separate away the cells which do not adhere to the substrate.
  • Candidate ligands which adhere to E, L or P-selectin, respectively, are determined via the labels on the cells. Such molecules are isolated, characterized, and their structure specifically identified.
  • Radiolabeled COS cells expressing cell surface E, L and/or P-selectin can be used as probes to screen com ⁇ pounds of the invention.
  • E, L or P-selectin transfected COS cells will adhere to a subset of compounds of the invention which can be resolved on TLC plates or adsorbed on PVC microliter wells. Adhesion tests are preferably done under physiological conditions. Adhesion to these compounds may require calcium, but will not be inhibited by heparin, chondroitin sulfate, keratin sulfate, or yeast phospho-mannan (PPME) .
  • PPME yeast phospho-mannan
  • Monosaccharide composition, linkage analysis, and FAB mass spectrometry of 5 of the purified compounds will indicate that the ligands for E, L or P-selectin share common structural characteristics which generally relate to the moieties A and B and the position in which they are held.
  • a complete cDNA for the E, L and/or P-selectin recep ⁇ tor 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 et al . , Proc. Natl Acad. Sci. USA (1987) 84 :8573) and the plasmid amplified in E. 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 E, L and/or P-selectin (Bevilacqua et al . , Science, (1989) 243 :1160; Polte et al . , Nucleic Acids Res (1990) 18.:1083; Hession et al . , Proc. Natl. Acad. Sci. USA (1990) jB_7:1673) . These publications are incorporated herein by reference for their disclosure of E-selectin and genetic material coding for its production.
  • compounds of formulae I and II may be adsor ⁇ bed to the bottoms of PVC microliter wells or resolved on TLC plates .
  • the compounds may be probed for their ability to support adhesion of E, L and/or P- selectm-transfected COS cells, untransfected COS cells, or COS cells transfected with a plasmid containing an irrelevant cDNA, under conditions of controlled detachment force (see Swank-Hill et al . , Anal. Biochem. (1987) 183:27: and Blackburn et al . , J. Biol. Chem. (1986) 261 :2873 each of which is incorporated herein by reference to disclose the details of such assaying methodology) .
  • NSAID or non-steroidal, anti-inflammatory drugs such as naproxen or lbuprofen which act as anti-inflammatory agents could be administered bound to the modified ligand and could be administered systemically in smaller amounts than usual while obtaining an equivalent effect or even greater anti-inflammatory effect at the site of inflamma- tion.
  • Any other drugs which might be attached include, but are not limited to, antibiotics, vasodilators and analgesics.
  • Such a drug delivery system would reduce any systemic effect normally caused by the drug in that the drugs could be administered in amounts of one-half to one- tenth the normal dose and still obtain the same anti- inflammatory result at the site of inflammation.
  • the invention compounds have considerable utility for the treatment of certain diseases, as set forth herein. However, this is not their only utility. Another utility is identification of particular chemical moieties that are responsible for, or contribute to ligand binding to the different selectins. Using the selectin binding assays described herein it is readily determined which chemical moieties that make up a selectin ligand contribute to selectin binding.
  • the compounds of the present invention can be used to treat a wide range of diseases, including diseases such as rheumatoid arthritis and multiple sclerosis.
  • the compositions of the invention should be 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.
  • the inflammation of rheumatoid arthritis is created when large numbers of white blood cells quickly enter the joints in the area of disease and attack the surrounding tissues.
  • 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 the E-selectin 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 thereby decreases the
  • 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 disease states which might be treatable using formulations of the invention include adult respiratory distress syndrome and various types of arthritis and asthma.
  • Radiolabeled compounds of the invention may be prepared in a sterile, non-pyrogenic medium and injected into the bloodstream of a patient at a dose to be determined in the usual way by the physician or radio- logist . After a sufficient period for a good balance to have been reached between (i) specificity of binding to activated endothelium compared to non-specific distri- bution and (ii) total amount of compound on activated endothelium, the compound is imaged in a conventional way, according to the nature of the label used. Use of radio- labelled compounds of the invention could be used to diagnose disease, such as the site of inflammation.
  • the compounds of the invention could also be used as laboratory probes to test for the presence of a selectin receptor such as a receptor of E, L and/or P-selectm in a sample.
  • a selectin receptor such as a receptor of E, L and/or P-selectm in a sample.
  • Such probes are preferably labeled such as with a radioactive label.
  • radioactive labeled atoms e.g. radioactive C, 0, N, P, or S
  • fluorescent dyes and enzyme labels which can be attached to compounds of the invention using known procedures. Labels as well as methods of attaching labels to sugar moieties are disclosed in U.S. Patent No. 4,849,513 issued July 18, 1989 to Smith et al . which patent is incorporated herein by reference to disclose labels and methods of attaching labels.
  • the compound of formulae I and II can be made using the general and specific synthesis schemes and examples described below. However, those skilled in the art will recognize variations thereof which are intended to be encompassed by the present invention. In general, the A and B moieties of formulae I and II are connected and held in a desired three-dimensional configuration.
  • the compounds of the present invention can be prepared in a number of ways. The following schemes show one preferred method of preparing the compounds of the present invention.
  • Scheme 1 shows the attachment of a B group to a naphthyl structure.
  • the chemistry depicted in Schemes 1-5 can also be used to functionalize the R 2 phenyl group and the aromatic positions on the flavanoid structure, R s , R 6 , R 7 , and R 8 .
  • Scheme 3 shows the Claisen Ireland rearrangement of the allylic ether to form a trisubstituted naphthyl structure where the second B group, "sugar 2 ,” is attached through an alkene linker.
  • Scheme 4 shows several methods of introducing functionality to a sugar. In all three products, the Sugar ! has been functionalized so that this B group acts as an acid moiety.
  • Scheme 5 shows another method of introducing acidic functionality onto a sugar .
  • Schemes 1-5 also apply to compounds where the naphthyl is replaced by a phenyl or flavanoid structure.
  • the compound shown above can then be reacted with a fluorescent probe, a multivalent compound, a ceramide, cholesterol or other lipid components, or a pharmaceutically active drug such as an anti-inflammatory drug.
  • a vast array of methods for carbon-carbon bond formation at the anomeric carbon are known in the art, which also can be applied to the formation of other heteroatom glycosides, as carbon-phosphorous, carbon-sulfur, carbon-nitrogen, or carbon-silicon bonds at the anomeric position which are understood to be within the invention.
  • the most common method for carbon-carbon bond formation at the anomeric carbon involves nucleophilic attack on this electrophilic center.
  • electrophilic sugars such as reducing sugars (or lactols) , alkyl glycosides, anomeric esters, glycosyl halides, imidates, anomeric trichloroacetimidates, glycals, lactones, thioglycosides, as well as oxygen-protected glycosides such as acetates and p-nitrobenzoates.
  • the carbon nucleophiles that have been used include silyl enol ethers, alkenes, allylsilanes, allylstannanes, cyanides, homoenolates, and organometallics such as Grignard reagents, organolithiums, cuprates, and aluminates. Further, procedures to synthesize carbon-glycosides based on metals (palladium, manganese, rhodium, and cobalt) have been developed. Concerted reactions such as [4+2] cycloadditions and sigmatropic rearrangements have also been employed to generate carbon glycosides.
  • glycosides useful for the generation of novel compounds provided by the present invention:
  • PG denotes a Protecting Group
  • Reagents efficient for the preparation of carbon glycosides include allyltrimethylsilane (Herscovici and Antonakis, 1992, Nat. Prod. Chem. 10:337; Postema, 1992, Tetrahedron 48:8545; Daves, 1990, Ace. Chem. Res. 23:201 Farmsell, 1985, Progress in Medicinal Chemistry 22 : 1 Hanessian and Pernet, 1976, Adv. Chem. Biochem. 33 :111 Carbohydrate Chemistry, Specialist Periodical Reports, Royal Chemical Society, 1968-1990, p. 1-24; preparation of allyl silanes: Anderson and Fuchs, 1987, Synthetic Commun. 27:621) and an array of carbon nucleophiles available from commercial sources.
  • Additional examples include, trimethylsilyl enol ethers, allyltrimethylsilane, E- and Z-crotyltrialkylsilanes, organoaluminum reagents, trialkylstannanes, propargylic trialkylstannanes,
  • [1- (acetoxy) -2-methyl-2-propenyl] -trimethylsilane reagent provides access to terminally oxygen substituted propenyl groups.
  • carbon glycosides can be produced in a few synthetic transformations, they do not necessarily form suitable carbon glycosides which could easily be used as alkylating agents for the preparation of novel carbohydrate mimics.
  • the present invention provides novel carbohydrate analogues for the preparation of carbohydrate mimetics.
  • Libraries of glycomimetics of complex carbohydrates such as, but not limited, to Sialyl Lewis x (sLe*) tetrasaccharide can be prepared (Rao et al .
  • halogen carbon glycosides are efficiently obtained from reaction of 2 -chloromethyl-3-trimethylsilyl -1 -propene or 2-chloromethyl-3-trimethoxysilyl-l-propene with an activated carbohydrate when the reaction is catalyzed by Lewis acid.
  • the allylsilanes can undergo a stereochemically controlled axial addition to the pyranose oxonium ions produced by Lewis acid catalysis and anomeric acetates.
  • Benzyl protected carbohydrates result in a stereoselective and efficient route to ⁇ -C-glycosides, incorporating an allylic chloride.
  • the tools and methods of the present invention are focused towards the incorporation of carbohydrates into existing and novel organic compounds, and combinatorial chemical libraries.
  • the compounds of the present inven ⁇ tion can be prepared individually or using combinatorial chemical libraries as described in W096/36627, which is incorporated herein by reference in its entirety.
  • the carbohydrate moieties employed for the generation of such flavanoid compounds and libraries include mono ⁇ mers, dimers, t ⁇ mers, oligomers, branched or unbranched, linked to a suitable functional group of a chemical moiety comprising such functional group.
  • Suitable functional groups include, but are not limited to, phenolic, hydroxyl, carboxyl, thiol, amido, and ammo groups
  • one or more such functional groups may be protected by suitable protecting groups during the coupling reaction.
  • suitable protecting groups include lower methyl-, benzyl-, benzoyl- , acetyl-, MOM, MEM, MPM, tBDMS, or TMS groups.
  • the pro ⁇ tecting groups may selectively be removed.
  • every molecule comprising at least one suitable functional group can be employed as substrate to react with the activated carbon glycosides/heteroatom glycosides of the present invention.
  • the monomers of the present invention i.e., the carbohydrates used for the formation of carbon glycosides, the carbon glycosides, and/or the substrates may have groups protecting part of the functional groups within the monomer. Suitable protecting groups will depend on the functionality and particular chemistry used to generate the novel compound or combinatorial chemical library. Examples for suitable functional protecting groups will be readily apparent to those of ordinary skill in the art, and can be found, among other places, in Greene and Wutz, 1991, Protecting Groups in Organic Synthesis, 2d ed., John Wiley & Sons, NY. Protecting groups typically used when modifying the anomeric position of carbohydrates are apparent to one of ordinary skill in the art. In addi ⁇ tion, a plurality of functional groups may be employed.
  • the C-atom of the carbohydrate used for the formation of the carbon glycosidic bond can be modified by differential protection of functional groups, as it will be apparent to those of ordinary skill in the art.
  • Most preferred pro ⁇ tecting groups of the present invention comprise benzyl- and acetyl-groups.
  • Carbon glycoside reagent can be functionalized to be used in a plethora of chemical reactions in order to form unique compounds. Suitable functionalized carbon glyco ⁇ sides can be attached, for example, to phenolic, hydroxyl, carboxyl, thiol, amino, amido, and/or equivalent func- tionality under mild conditions. Activated forms of C- glycosides are alkylhalide, alkenyl halide, or some equivalent activated form. The coupling reactions for activated C-glycosides can be performed to form novel compounds under standard conditions typically used for alkyl and alkenyl chlorides, bromides, iodides, acetates, alcohols, Grignards, etc.
  • alkenyl C- glycosides offer routes into Cope rearrangements, Claisen rearrangements, and allylic couplings, as they are readily known by those of ordinary skill in the art and as are described in various examples provided hereinbelow to form novel glycomimetics and unique compounds.
  • alkenyl C-Glycoside compounds described and utilized here are of particular utility due to the reactivity of the activated alkenyl C-glycoside reagent, and the diverse array of transformations and functional group modifications possible around the alkenyl moiety.
  • the scheme below illustrates some of the more common transformations that can be applied to these structures, with the understanding that many additional modifications are possible to one of ordinary skill in the art.
  • the alkenyl moiety is easily converted to the saturated substituted alkyl form using standard catalytic hydrogenation conditions as found in advanced organic chemistry reference textbooks.
  • the vicinal diol derivative can be prepared by oxidation with osmium tetraoxide and NMO or similar bishydroxylation conditions as described in standard texts on organic chemistry.
  • the ketone derivative can be made from diol by oxidative cleavage reaction conditions including periodate oxidation. Alternately, the ketone can be prepared by direct treatment of the alkene with osmium tetroxide and periodate. The ketone can then provide the secondary alcohol by reduction with reducing agents such as sodium borohydride.
  • the diol group described above is suited to a variety of O-alkylation and O-acylation reactions, with O- sulfation being a particularly useful method for introducing anionic functionality into the linker.
  • the ketone can undergo subsequent modifications via wittig reactions as described to yield the substituted alkene groups, or following reduction by catalytic hydrogenation, yielding substituted alkyl linker groups.
  • the common transformations described above are generally known to one skilled in the art and the methodologies described, and equivalent methodologies are available in advanced organic chemistry textbooks, and references cited therein. For some general references to these chemistries please see the appropriate Example.
  • the rearrangement of allyl aromatic ethers to ortho or para allylaromatic alcohols, or the rearrangement to adjacent positions relative to the alkylated aromatic alcohol moiety can be accomplished in a two step format.
  • the first step is to alkylate the aromatic alcohol, which can be done using the procedure herein disclosed or by using the optional procedure used in the following example using sodium methoxide.
  • the result is the o-alkylated aromatic alcohol.
  • the alkylated aromatic alcohols can then be refluxed in the appropriate solvent such as dimethylaniline or 1,2- dichlorobenzene for 3-12 hours (depending on the substrate) to give the Claisen-Ireland product.
  • appropriate solvent such as dimethylaniline or 1,2- dichlorobenzene for 3-12 hours (depending on the substrate) to give the Claisen-Ireland product.
  • the rearrangement of allyl phenyl ethers to ortho or para allyl phenols can be accomplished in a two step format.
  • the first step is to alkylate the phenol, which can be done using the procedures herein disclosed or by using the optional procedure used in the following example using sodium methoxide.
  • the compounds of the invention such as various ligands of structural formulae I and II can be administered to a subject in need thereof to treat the subject by either prophylactically preventing inflammation or relieving it after it has begun.
  • the ligands 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 a liquid salt solution carrier.
  • the formulation of choice can be accomplished using a variety of excipients includ ⁇ ing, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin cellulose, magnesium carbonate, and the like.
  • Oral compositions may be taken the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders. Particularly useful is the administration of the compounds directly in transdermal formulations with permeation enhancers such as DMSO. Other topical formulations can be administered to treat dermal inflammation.
  • 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 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.
  • the ligand molecules of the invention can be formulated in suppositories and, some cases, aerosol and tranasal compositions.
  • the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides.
  • suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%.
  • 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 formula ⁇ tions 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.
  • the compounds of the instant invention may also be administered as injectables.
  • m ectable compositions are prepared as liquid solutions or sus- pensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.
  • Compounds of formulae I and II can be mixed with compatible, pharmaceutically acceptable excipients. 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.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those of ordinary skill in the art. See, e.g. Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985.
  • the composition or formulation to be administered will, in any event, contain a quantity of the compounds adequate to achieve the desired state in the subject being treated.
  • the various compounds of the present invention can be used by themselves or in combination with pharmaceutically acceptable excipient materials as described above.
  • the compounds of the invention can be made as conjugates wherein they are linked in some manner (e.g., via the R : moiety) to a label.
  • the compounds can act as biochemical delivery systems for the label so that a site of disease can be detected.
  • carbohydrates can be labelled by a variety of procedures, for example: esterification of hydroxyl bonds to form a structure capable of complexing directly with a radioisotope or NMR enhancer; reaction of the carbohydrate with amino diacetic acid (IDA) in organic solvent to form an N-linked g -oside derivative which would be capable of complexing w a radioisotope via the nitrogen and oxygen atoms of the IDA group; or coupling of the carbohydrate to amino acids which may be labelled directly (e.g. cysteine, tyrosine) or labelled via a bifunctional chelating agent (e.g., lysine) .
  • IDA amino diacetic acid
  • radioactive atoms would include, for example, technetium 99m ( 99m Tc) , iodine-123 ( 123 I) or indium- Ill ( l ⁇ In) for scintigraphic studies, or for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI) , a label such as gadolinium, manganese or iron, or a positron-emitting isotope such as iodine-124, fluorine-19, carbon-13, nitrogen-15 or oxygen- 17.
  • 99m Tc technetium 99m
  • iodine-123 123 I
  • indium- Ill l ⁇ In
  • NMR nuclear magnetic resonance
  • MRI nuclear magnetic resonance
  • a label such as gadolinium, manganese or iron
  • a positron-emitting isotope such as iodine-124, fluorine-19, carbon-13, nitrogen-15 or oxygen- 17.
  • THF Tetrahydrofuran
  • DMF dimethylformamide
  • the reactions set forth below are done generally under a positive pressure of nitrogen or with a drying tube, at ambient temperature (unless otherwise stated) , in anhydrous solvents, and the reaction flasks were fitted with rubber septa for the introduction of substrates and reagents via syringe. Glassware was oven dried and/or heat dried. Analytical thin layer chromatography (TLC) was performed on glass-backed silica gel 60 F 254 plates Analtech (0.25 mm) and eluted with the appropriate solvent ratios (v/v) which are noted where appropriate. The reactions were assayed by TLC and terminated as judged by the consumption of starting material.
  • TLC thin layer chromatography
  • Hydrogenolysis can be done at the pressure indicated in the examples, or at ambient pressure.
  • solvents can include but not restructed to, methanol, ethyl acetate, ethanol, acetic acid or combinations thereof.
  • methanol with a catalytic amount of acetic acid or ethyl acetate and methanol can be used as the hydrogenation solvent.
  • 5% or 10% palladium on carbon (1 g for every 50 grams of starting material with the catalyst wetted with toluene under argon) is evacuated and hydrogen gas is added and the process repeated three times. The reaction is shaken or stirred for several hours until the deprotection is complete.
  • the reaction can be done under ambient pressures or can be done a hydrogenation bomb at appropriate pressures (generally 10-
  • the reaction is terminated by removal of the excess hydrogen gas, flushing the reaction vessel with an inert atmosphere and then filtering the contents through Celite to remove the catalyst and washing the catalyst with 30% methanol in chloroform or appropriate solvent system. Concentration in vacuo afforded the desired compound.
  • the product can be purified by column chromatography using Baker grade fresh silica gel (47- 61mm) and a suitable solvent system. For example, 10% ethyl acetate in hexanes and then with 30% ethyl acetate in hexanes. The silica gel is eluted with methanol and checked by TLC for any product material . The solvents are removed in vacuo and the product dried under vacuum. The desired product is recovered.
  • 9-BBN For a compound containing an alkene, (1.00 mmole equiv. ) is dissolved in an appropriate solvent suitable for the compound to be reduced with 9-BBN (9-borabicyslo
  • Solvents can include but not restricted to, tetrahydrofuran (THF) , hexanes and diethyl ether, or combinations thereof.
  • THF tetrahydrofuran
  • 9-BBN 9-BBN pre-dissolved in THF (1.00 mmole equiv.
  • the reaction contents are stirred at 0°C and the cooling bath (water/ice) is allowed to melt the reaction allowed to stir at ambient temperature for 8 hours or until the reaction is complete via analysis by TLC.
  • the reaction is terminated by the careful addition acetone and stirred for 1 hour at room temperature.
  • the reaction contents are cooled to 0°C and then 1.0 M NaOH and 30% H202 are added to decompose the alkyl borate.
  • the contents are stirred until the alkyl borate decomposes to the carbonol .
  • An extraction solvent such as chloroform or ethyl acetate is added and the heterogeneous layers are separated and the organic phase is washed with l.OM hydrochloric acid, water and brine.
  • the washed product is dried over anhydrous sodium sulfate and filtered.
  • the product can be purified by column chromatography using Baker grade flash silica gel (47- 61mm) and a suitable solvent system.
  • the reaction can be assayed by TLC or an aliquot of the reaction acetate. The aliquot is checked by -NMR. The reaction is terminated by the careful addition sodium bisulfite (contains a mixture of NaHS0 3 and Na 2 S 2 0 5 ) , stirred for 1 hour at room temperature and then water An extraction solvent such as chloroform is added and the heterogeneous layers are separated and the organic phase is washed with l.OM hydrochloric acid, water and brine. The washed product is dried over anhydrous sodium sulfate and filtered. The product can be purified by column chromatography using Baker grade flash silica gel (47- 61mm) and a suitable solvent system.
  • the hydroxy groups of the target molecule are sulfated under standard conditions: For example, to a solution of 0.35 g 3,4-di-C-fucosyl caffeic acid (0.603 mmole, 1 mmole equiv.) in 7.2 mL pyridine was added 1.15 g sulfur trioxide pyridine complex (7.24 mmole, 12 mmole equiv.) and the reaction was stirred at ambient temperature for 12 hours. The reaction was complete as assayed by TLC at CHC1 3 :MeOH:H 2 0 10:10:1 (v/v) as assay conditions.
  • Ketone Formation Oxidation of the alkene to the ketone via catalytic oxidation with osmium tetroxide sodium periodate.
  • osmium tetroxide sodium periodate To a stirred solution of the olefin, (1.00 mmole equiv.) in 1% water in acetone (0.5M) at 0°C is added osmium tetroxide pre-dissolved acetone (0.01 mmole equiv.) and sodium periodate (2.00 mmole equiv.) is carefully added as a solid.
  • the reaction contents are stirred at 0°C and the cooling bath (water/ice) is allowed to melt and the reaction allowed to stir at ambient temperature for 18 hours or until the reaction is complete via analysis by TLC.
  • the reaction can be assayed by TLC or an aliquot of the reaction contents is removed, quenched into aqueous sodium etasulfite and extracted with ethyl acetate. The aliquot is checked by L H-NMR.
  • the reaction is terminated by the careful addition sodium bisulfite (contains a mixture of NaHS0 3 and NA 2 S 2 0 5 ) , stirred for 1 hour at room temperature and then water.
  • An extraction solvent such as chloroform is added and the hydrochloric acid, water and brine.
  • the washed product is dried over anhydrous sodium sulfate and filtered.
  • the product can be purified by column chromatography using Baker grade flash silica gel (47-61mm) and a suitable solvent system.
  • a compound containing a ketone (1.00 mmole equiv.) is dissolved in an appropriate solvent suitable for the compound to be reduced with sodium borohydride or equivalent.
  • Solvents can include but not restricted to, tetrahydrofuran (THF) , hexanes and diethyl ether, or combinations thereof.
  • THF tetrahydrofuran
  • hexanes and diethyl ether or combinations thereof.
  • sodium bobohydride pre-dissolved/suspended in THF (1.00 mmole equiv.) is carefully added.
  • reaction contents are stirred at 0 * C and the cooling bath (water/ice) is allowed to melt the reaction allowed to stir at ambient temperature for 8 hours or until the reaction is complete via analysis by tic.
  • the reaction is terminated by the careful addition acetone and stirred for 1 hour at room temperature. Water is carefully added and the reaction contents stirred to decompose the alkyl borate. The contents are stirred until the alkyl borate decomposes to the carbonol .
  • An extraction solvent such as chloroform or ethyl acetate is added and the heterogeneous layers are separated and the organic phase is washed with l.OM hydrochloric acid, water and brine. The washed product is dried over anhydrous sodium sulfate and filtered.
  • the product can be purified by column chromatography using Baker grade flash silica gel (47-61mm) and a suitable solvent system. For example, 10% ethyl acetate in hexanes and then with 30% ethyl acetate in hexanes or appropriate solvent mixtures dependent upon the particular substrate used.
  • the silica gel is eluted with methanol and checked by TLC for any product material . The solvents are removed in vacuo and the product dried under vacuum the desired product is recovered.
  • the mmole equivalents refers to the reaction substrate to be functionalized by the reaction with the carbon glycoside reagent per position to be alkylated. Additional functional group transformations can be accomplished by the skilled artisan using standard reaction conditions. For example, the transofmration of allylic halides into allylic amines can be via the allylic azide with reduction of the azide to the amine with triphenylphosphine in water. The amine is then available for amide bond formation.
  • reaction contents are warned to for a period of 2 hours; carefully watching for the evolution of hydrogen.
  • the reaction contents are stirred using a mechanical stirrer while being gently refluxed for a period of 1.5 hours.
  • a benzyl protected carbon gylcoside reagent to be used (1.50 mmole equiv.) is slowly added dropwise in anhydrous tetrahydrofuran (total reaction concentration of 0.2 to 0.5 M) over a period of 1-2 hours and stirred for 4 hours.
  • reaction contents are removed and quenched into l.OM HCl and extracted with ethyl acetate; the TLC conditions used are 5% methanol in chloroform (v/v.]
  • the reaction is then diluted with tolune and terminated by the careful addition of 50% methanol in toluene at 0 * C to consume the residual sodium hydride, follwed by acidification of l.OM hydrochloric acid until the pH is about 2.
  • the reaction contents are diluted with ethyl acetate.
  • the hetero- geneous layers are separated and the organic phase is washed twice with portions of l.OM hydrochloric acid, saturated sodium thiosulfate and brine.
  • the product can be purified by column chromatography using Baker grade flash silica gel (47-61 mm) and a suitable solvent system. For example, 10% ethyl acetate in hexanes and then with 30% ethyl acetate in hexanes. The silica gel is eluted with methanol and checked by TLC for any product material . The solvents are removed in vacuo and the product dried under vacuum. The desired product is recovered. General alkylation conditions using Cesium Carbonate as a mild base for the alkylation of thiols, amines, carboxylic acids and the like.
  • the product is dried over anhydrous sodium sulfate and filtered to remove the drying agent.
  • the solvent is removed in vacuo.
  • the product can be purified by column chromatography using Baker grade flash silica gel (47-61mm) and a suitable solvent system. For example, 10% ethyl acetate hexanes and then with 30% ethyl acetate in hexanes.
  • the silical gel is eluted with methanol and checked by TLC for any product material .
  • the solvents are removed in vacuo and the product dried under vacuum. The desired product is recovered. Removal of the acetyl protecting groups from the acetylated carbon glycosides.
  • This crude product was heated with sodium iodide (3 g) in acetone (40 ml) at 450 * C for 2 hours. After cooling, the mixture was evaporated and the residue worked up with water (50 ml) and ethyl acetate (200 ml) . The crude mixture was purified on a silica gel column (hexane ethyl acetate, 2:1) to provide the iodide 2 (2.9 g, 76%) .
  • a preferred method for the synthesis of terminally substituted halogen carbon glycosides comprises a chemical reaction of 2-chloromethyl-3-trimethylsilyl-1-propene (SAF Bulk Chemicals) , or 2-chloromethyl-3-trimethoxysilyl-l- propene (SAF Bulk Chemicals) , or 2-chloromethyl-3- trimethoxysilyl-1-propene by Gelest Inc. (U.S. Patent No.
  • the product was purified either by crystallization in methanol at 0°C, or by column chromatography using Baker grade flash silica gel (47-61mm) (ratio of 20 to 1) , followed by elution with 5 to 10% ethyl acetate in hexanes giving a white solid (98%) , mp 47-49°C.
  • the a- to ⁇ - ratios of the C-glycosidation reaction was > 10:1 in favor of the ⁇ -fucose derivative (Cha, et al . , 1982, J. Am. Chem. Soc. .104.:4976) .
  • the reagent ratios for the remaining per- O-acetylated carbohydrates were for example: 1,2,3,4,6- penta-O-acetyl-D-galactopyranoside (1.00 mmole equiv. ) and 2-chloromethyl-3-trimethylsilyl-1-propene (2.00 mmole equiv.) were dissolved in acetonitrile (1.3M) . Boron trifluoride etherate (2.00 mmole equiv.) and trimethyl- silyltrifluoromethane sulfonate (0.40 mmole equiv.) were carefully added neat at room temperature. The reaction was refluxed for 6 hours and worked up as described. TLC 30% ethyl acetate in hexanes.
  • l-O-acetyl-2 , 3 , 4 , 6- tetra-O-benzyl-D-galactopyranose were subjected to the same reaction conditions as have been described for L- fucopyranoside, resulting in the ⁇ -C-glycosides of galactose (5) (84%, oil) , as reflected below:
  • the reaction contents were concentrated to half of the original volume of THF, poured into cold water and then extracted with EtOAc. The organic layer was washed twice with water, l.OM HCl and again with water. The product was dried over anhydrous sodium sulfate and filtered to remove the drying agent. The solvent was removed in vacuo which afforded a light yellow solid. The product was dissolved in methanol and then concentrated in vacuo at low temperatures twice to remove any residual solvents. The product was dissolved in 150 ml warm methanol and cooled to 0°C overnight. Filtration of the solids results in 40.8 grams as a white crystalline solid. Concentration of the mother liquors to half of the original volume and again cooling to 0°C overnight gave an additional 10.87 grams of a white crystalline solid.
  • the reagent ratios for the remaining per- O-acetylated carbohydrates were for example: 1,2, 3,4, 6-penta-O-acetyl-D-galactopyranoside (1.00 mmole equiv.) and 2-chloromethyl-3-trimethylsilyl-l-propene (2.00 mmole equiv.) were dissolved in acetonitrile (1.3M) .
  • Boron trifluoride etherate (2.00 mmole equiv.) and trimethylsilyltrifluoromethane sulfonate (0.400 mmole equiv.) were carefully added neat at room temperature. The reaction was refluxed for 6 hours and worked up as described. TLC 30% ethyl acetate in hexane. '
  • the reaction was terminated by cooling to room temperature, pouring the reaction contents on 100 ml of water, followed by extraction of the crude o;-C-glycoside with ethyl acetate.
  • the heterogenous layers were separated and the organic phase was washed with portions of water, saturated sodium bicarbonate, l.O M hydrochloric acid and brine.
  • the crude extract was dried over anhydrous sodium sulfate, filtered, and the solvent was removed in vacuo to afford an oil that was purified by column chromatography on Baker grade flash silica gel (47- 61mm) (ratio of 20 to 1) , eluted with 10% ethyl acetate in hexanes.
  • the major compound was the 2, 3,4-tri-O-acetyl- ⁇ -C-L-fucopyranoside (85%), and a minor compound was a small amount of starting material.
  • the baseline material observed by TLC probably representing a third unidentified molecule.
  • the deprotection of the fucose reagent was done in methanol with a catalytic amount of sodium metal added to the stirring methanol .
  • the reaction was terminated by the careful addition of l.OM HCl until the pH was approximately 2.
  • the solvent was removed in vacuo .
  • the deprotected fucose is reflected in the following structure:
  • reaction conditions used for the ⁇ -C-glycosid- ation of 1, 2 , 3 , 4-tetra-O-acetyl-L-fucopyranoside were applied to 1, 2, 3 , 4 , 6-penta-O-acetyl-D-glycopyranose, and yielding the expected ⁇ -C-glycosides of o-C-glucose (20%) .
  • Example 13 1-Deoxy- ⁇ -l-Acetylenyl-l, 2.4-tri-O-Benzyl-L-Fucose (13)
  • Fucose-Phenylalanine Amide (14) Carbonyl diimidazole (2.62 g, 16.14 mmol) was added to a solution of m-iodobenzoic acid (4g, 16.14 mmol) in tetrahydrofuran (100 ml) , the mixture was stirred at room temperature (45 min) . Phenylalanine benzyl ester p- toluenesulfonate (6.90 g, 16.14 mmol) was added, the mixture was stirred (18), and concentrated to dryness.
  • Triethylamine (0.73 ml, 5.26 mmol) was added followed by tetrakistriphenyl-phosphine palladium (100 mg) and copper (1) iodide (40 mg) .
  • the reaction was stirred at room temperature (6h) , diluated with water (100 ml) , and extracted with ethyl acetate (3X20 ml) .
  • the combined organic phases were washed with IN aq. HCl (40 ml) , saturated aqueous sodium bicarbonate (40 ml) and brine (40 ml) , dried (MgSO and concentrated.
  • the residue was purified on silica gel (20% ethyl acetate/hexane) to give the coupled product (0.6g, 28% yield) .
  • the coupled product (0.6 g, 0.75 mmo) was dissolved in ethyl acetate (5 ml) .
  • 10% Palladium/carbon (0.6 g) was suspended in methanol (40 ) and the ethyl acetate solution was added via cannula. After stirring under hydrogen for 4 days, the mixture was filtered through Celite and the filtrate was concentrated to dryness giveing the title compound 14 (0.24 g, 72% yield) .
  • reaction mixture is separated on silica gel giving the desired product .
  • Sodium metal (4 spheres) are washed in hexane and added to anhydrous methanol (20 mL) .
  • 0.10 mL of the resulting sodium methoxide solution is added to a solution of the product from example 20 in anhydrous methanol (10 L) .
  • the reaction is concentrated to dryness giving ' the desired product .
  • the product from example 21 is dissolved in methylene chloride/dimethyl formamide (20/1) and TBDMS-Cl (0.53 g, 3.51 mmoles) is added followed by imidazole (0.34 g, 5.02 mmoles) . After stirring at room temperature for 20 hours, TBDMS-Cl (0.53 g, 3.51 mmoles) and imidazole (0.34 g, 5.02 mmoles) are added. Stirring is continued for an additional 20 hours after which, the reaction is quenched with methanol (10 mL) . After stirring for 30 minutes at room temperature, the reaction is concentrated to dryness and the product is purified on silica gel.
  • the product from example 22 is dissolved in pyridine and an equivalent volume of acetic anhydride is added. After stirring at room temperature for three days, the reaction is concentrated to dryness and the residue is dissolved in ethyl acetate. After washing with IN HCl (3 X), saturated aqueous sodium bicarbonate (6 X) , water, saturated aqueous copper sulfate (2 X) , and brine (10 mL) , the organic phase is dried over anhydrous magnesium sulfate, filtered and concentrated to dryness giving the desired product (1.98 g, 81%) .
  • Example 27 Sodium metal (4 spheres) are washed in hexane and added to anhydrous methanol (20 mL) . 0.10 mL of the resulting sodium methoxide solution is added to a solution of the product from example 25 in anhydrous methanol . After stirring at room temperature for 24 hours, the reaction is concentrated to dryness giving the desired product .
  • Example 27
  • the product described m example 26 is dissolved in methanol and an equivalent volume of 2N aqueous sodium hydroxide (4 equivalents) is added. The reaction is stirred at room temperature for 20 hours and neutralized with methanol washed Amberlyst acidic ion exchange resin. Filtration and concentration of the resulting solution provides the desired product.
  • Example 29 Sodium metal (4 spheres) were washed in hexane and added to anhydrous methanol (20 mL) . 0.20 mL of the resulting sodium methoxide solution is added to a solution of the product from example 17 in anhydrous methanol . After sr-ir ⁇ ng at room temperature for 24 hours, the reaction is neutralized with amberlite acidic ion exchange resin. After removal of the resin by filtration, the filtrate is concentrated to dryness giving the desired product .
  • Example 29
  • This product is prepared from the product of example 30 using methodology similar to that used in example 25.
  • This product is prepared from the product of example 31 using methodology similar to that used in example 28.
  • This product is prepared from the product of example 32 using methodology similar to that used in example 27.
  • This product is prepared from the product of example 34 using methodology similar to that used in example 20.
  • This product is prepared from the compound described in example 44 using similar conditions to those described in example 21.
  • This product is prepared from the product of example 51 using methodology similar to that used in example 46.
  • This product is prepared from the product of example 52 using methodology similar to that used in example 49.
  • This product is prepared from the product of example 53 using methodology similar to that used in example 48.
  • This product is prepared from the product of example 55 using methodology similar to that used m example 40.
  • the product from example 59 is dissolved in methylene chloride/dimethyl formamide (20/1) and TBDMS-Cl (0.53 g, 3.51 mmoles) is added followed by imidazole (0.34 g, 5.02 mmoles) . After stirring at room temperature for 20 hours, TBDMS-Cl (0.53 g, 3.51 mmoles) and imidazole (0.34 g, 5.02 mmoles) are added. Stirring is continued for an additional 20 hours after which, the reaction is quenched with methanol (10 mL) . After stirring for 30 minutes at room temperature, the reaction is concentrated to dryness and the product is purified on silica gel.
  • This product is prepared from the compound described m example 61 using similar conditions to those described m example 24.
  • Example 65 The product of Example 65 is deprotected according to Example 49.
  • the ketal is then prepared using the method described in Example 50.
  • the ketal and 1-deoxy-l-a- (3- chloromethallyl) -2,3, 4-tr ⁇ -0-acetylfucose are dissolved in dimethylformamide and potassium carbonate is added followed by tetrabutyl ammoniumiodide.
  • the reaction is diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate (3 X) and brine (2 X) .
  • the mixture is dried over anhydrous magnesium sulfate, filtered and concentrated.
  • the residue is purified on silica gel to give the desired product.
  • This product is prepared from the product of example 66 using methodology similar to that used in example 63.
  • This product is prepared from the product of example 67 using methodology similar to that used in example 63.
  • This product is prepared from the product of example 68 using methodology similar to that used m example 65.
  • the product from example 69 is dissolved in tetrahydrofuran and an equivalent volume of 6N hydrochloric acid is added. After stirring at room temperature for 24 hours, the reaction is concentrated to dryness to give the desired product.
  • This product is prepared from the product of example 70 using methodology similar to that used in example 58.
  • the product from example 77 is dissolved m methylene chloride/dimethyl formamide (20/1) and TBDMS-Cl (0.53 g, 3.51 mmoles) is added followed by imidazole (0.34 g, 5.02 mmoles) . After stirring at room temperature for 20 hours, TBDMS-Cl (0.53 g, 3.51 mmoles) and imidazole (0.34 g, 5.02 mmoles) are added. Stirring is continued for an additional 20 hours after which, the reaction is quenched with methanol (10 mL) . After stirring for 30 minutes at room temperature, the reaction is concentrated to dryness and the product is purified on silica gel.
  • This product is prepared from the compound described in example 79 using similar conditions to those described m example 24.
  • Example 85 Sodium metal (4 spheres) are washed m hexane and added to anhydrous methanol (20 mL) . 0.10 mL of the resulting sodium methoxide solution is added to a solution of the product from example 74 in anhydrous methanol . After stirring at room temperature for 24 hours, the reaction is concentrated to dryness giving the desired product .
  • Example 85
  • This product is prepared from the product of example 85 using methodology similar to that used in example 81.
  • This product is prepared from the product of example 4o using methodology similar to that used in example 4k.
  • This product is prepared from the product of example 88 using methodology similar to that used in example 76.
  • the aromatic hydroxyl groups of flavones are alkylated under standard conditions.
  • the hydroxy groups of di- C-fucosyl flavones are sulfated under standard conditions to provide persulfated di-C-fucosyl flavones.
  • Example A Selectin Binding An ELISA assay was employed that uses recombinant fusion proteins composed of extracellular portions of the human selectins joined to human immunoglobulin heavy chain CH 3 , CH 2 , and hinge regions. See, for example, Walz et al . , Science (1990) 250:1132 ,* Aruffo et al . , Cell (1991) 6_7:35; Aruffo et al . , Proc. Natl. Acad. Sci. USA (1992) JL9:2292.
  • the assay is well known in the art, and generally consists of the following three steps:
  • step I This solution was then placed in the microliter wells that were prepared in step I.
  • the plate was incubated at 37°C for 45 minutes to allow the soluble receptor to bind to its natural ligand. In the presence of a strong inhibitor only a few receptors should be free to bind to the microliter plate coated with the natural ligand.
  • the positive control is the signal produced by the soluble receptor when it is allowed to react with 2,3 sLe x glycolipid in the microliter wells m the absence of any inhibitor. This was considered 100% bmding.
  • the signal produced by the receptor that had been previously treated with an inhibitor was divided by the signal produced by the positive control and multiplied by 100 to calculate the % receptor bound to the well, or the percent of control binding.
  • Tables A and B lists the extent to which the invention compounds inhibit binding of E, L and P-selectin to 2,3 sLe x glycolipid terms of IC 50 values.
  • ligands described above could be obtained by selectins more rigid spacers in order to maintain the appropriate statistical average distance between the sialic acid and fucose moieties in space thereby improving the inhibitory property of such structures towards the selectins.
  • Further modifications of these compounds e.g., attaching them through chemical linkages on appropriate molecular supports and use of analogs or derivatives of sialic acid and L-fucose are also considered to be within the scope of the present invention.
  • the cells were incubated for 15-30 mins on ice, washed twice with staining medium, and resuspended 0.5 ml of staining medium before flow cytometric analysis on a FACScan (Becton Dickinson & Co., Mountain View, CA) .
  • FACScan Becton Dickinson & Co., Mountain View, CA
  • the staining was also done in the presence of 10 mM EGTA.
  • protease sensitivity and the requirement for sialic acid of this interaction HL-60 cells in D-PBS and 1% BSA were incubated with either trypsin or Arthrobacter or Clost ⁇ dium sialidases at 37°C before resuspend g in staining medium.
  • 50 ⁇ g/ml fucoid 50 ⁇ g/ml fucoid
  • the invention compounds would be used to prevent or treat sepsis.
  • the procedure would consist of using two administration routines for each of the compounds tested wherein they are delivered in physiological saline.
  • first between 1 and 10 mg of compound per kg of body weight is administered in three separate doses at 24, 22, and 21 nours before a lethal challenge of bacteria
  • compound can be administered in a single dose simultaneously with tne bacterial challenge. In both instances the compounds would considerably extend the liefetime of the baboons that receive the multiple or single dose treatment and they would survive well beyond 48 hours.
  • the compounds of the invention are useful for treating diseases, preferably diseases that have an inflammatory component-Adult Respiratory Distress Syndrome (ARDS) , ischemia and reperfusion injury, including strokes, mesenteric and peripheral vascular disease, organ transplantation, and circulatory shock (in this case one or many organs might be damaged following restoration of blood flow) .
  • ARDS inflammatory component-Adult Respiratory Distress Syndrome
  • ischemia and reperfusion injury including strokes, mesenteric and peripheral vascular disease, organ transplantation, and circulatory shock (in this case one or many organs might be damaged following restoration of blood flow) .

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Abstract

L'invention concerne des composés qui possèdent une activité de liaison avec la sélectine. Ces composés présentent une configuration stable d'un point de vue tridimensionnel pour l'acide sialique et la fucose, ou des analogues, des dérivés ou des mimétiques de ces groupes, de telle sorte que l'acide sialique et le fucose ou leurs mimétiques sont séparés par un segment de liaison qui permet la liaison entre ces groupes et les sélectines. Ces composés sont représentés par les formules générales (I) et (II).______
PCT/US1997/002951 1996-02-21 1997-02-20 MIMETIQUES DE SIALYL LEWISx CONTENANT DES SQUELETTES DE FLAVANOIDE WO1997031007A1 (fr)

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AU19752/97A AU1975297A (en) 1996-02-21 1997-02-20 Sialyl lewisx mimetics containing flavanoid backbones
EP97907858A EP0888369A1 (fr) 1996-02-21 1997-02-20 Mimetiques de sialyl lewis x contenant des squelettes de flavanoide

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Publication number Priority date Publication date Assignee Title
US8481590B2 (en) 2010-02-12 2013-07-09 N30 Pharmaceuticals, Inc. Chromone inhibitors of S-nitrosoglutathione reductase
US8669381B2 (en) 2010-02-12 2014-03-11 N30 Pharmaceuticals, Inc. Chromone inhibitors of S-nitrosoglutathione reductase
US8759548B2 (en) 2010-02-12 2014-06-24 N30 Pharmaceuticals, Inc. S-nitrosoglutathione reductase inhibitors
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