CA2138645A1 - Substituted lactose derivatives as cell adhesion inhibitors - Google Patents

Abstract

Compounds and methods of making them having formula (1) are described which bind to selectin receptors and thus mod-ulate the course of inflammation, cancer and related diseases by modulating cell-cell adhesion events. In formula (1), R1 is inde-pendently H or lower alkyl(1-4C); R2 is H, OH or lower alkyl(1-4C), alkylaryl or one or more additional saccharide resi-dues; R3 is a negatively charged moiety including S04--, P04--, or related group; Y is H or lower alkyl(1-4C); and X is -CHR4(CHOR1)2CHR50R1 wherein R4 and R5 are each independently H, lower alkyl(1-4C), or taken together result in a five- or six-membered ring optionally containing a heteroatom selected from the group consisting of O, S, and NR1; said five- or six-membered ring optionally substituted with one substituent selected from the group consisting of Rl, CH20R1, OR1, OOCR1, NR12, NHCOR1, and SR1 with the proviso that if X represents a hexose substituent R3 and R4, taken togeth-er, cannot provide a hexose substituent.

Description

WO 94/00477 213 8 6 ~ 5 PCI/US93/06110 SUBSTITUTED LACTOSE DERIVATIVES
AS CELL ADHESION INHIBITORS

Technical Field The invention relates to compounds useful in the tre~tment of infl~mm~tion, allergic reactions, autoimmnnP tli~e~ces~ and related conditions. More specifically, the invention conr~-rnc substituted lactose that binds to selectin receptors and to ph~rrn~reutical compositions cont~ining them. The present invention is also directed to synthetic methods useful in obtaining these analogs and other lactose derivatives.

Back~round Art It is now well established that cellular 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 lig~nrlc One class of receptors that recognize carbohydrate-based ligands me~ t~s the adhesion of circulating neutrophils to stimulated vascular endothelium. This is a primary event of the infl~mm~tQry response and appears to be involved as well in allergic and autoimmnn~ responses. Several receptors have been implicated in this interaction, including a family of putative lectins that includes gpgoMEL (Leu8), ELAM-l, and GMP-140 (PADGEM) and (Gong, J.-G., et al., Nature (1990) 343:757; Johnston, G.I., et al., Cell (1989) 56:1033; Geoffrey, J.S., and Rosen,S.D., J. Cell Biol. (1989) 109:2463; Lasky, L.A., et al., Cell (1989) 56:1045).
These lectins have been termed L-SELECTIN, E-SELECTIN, and P-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-l or TNF (Bevilacqua, M.P., et al., Science (1989) 243:1160). Thetime 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. USA (1987) WO 94/00477 PCr/US93/06110 2~ 3~
84:9238) have demonstrated that human neutrophils or HL-60 cells will adhere to COS cells transfected with a plasmid cont~ining a CDNA encoding for the E-SELECTIN receptor. Information regarding the DNA sequences encoding for endothelial cell-leukocyte adhesion molecules are disclosed within PCT publishedapplication WO90/13300 published November 15, 1990.
Recently, several different groups have published papers regarding the ligand for E-SELECTIN. Lowe et al., (1990) Cell, 63:475-484 reported a positive correlation between the E-SELECTIN dependent adhesion of HL-60 cell variants and tr~nefected cell lines, with their expression of the sialyl Lewis x (sLex) oligosaccharide, Neu Nac oc2-3Gal-~1-4(Fuc oc1-3)-GlcNAc. By trancfecting cells with plasmids cont~ining an oc(1,3/1,4) fucosyltransferase, they were able to convert non-myeloid COS or CHO lines into sLex-positive cells that bind in an E-SELECTIN dependent m~nn~r Attempts to block E-SELECTIN dependent adhesion using anti-sLex antibodies were uninterpretable due to the ag~ tin~tiQn of the test cells by the antibody. They concluded that one or more members of a family of oligosacch~r~dec consisting of sialylated, fucosylated, lactos~minQglycans are the ligands for the lectin domain of E-SELECTIN. Phillips et al., (1990) Science, 250: 1130-1132 used antibodies with reported specificity for sLex to inhibit the E-SELECTIN dependent adhesion of HL-60 or LEC11 CHO cells to activated endothelial cells. Liposomes cont~ining difucosylated glycolipids withterminal sLex structures inhibited adhesion, while those cont~ining nonsialylated Lex structures were partially inhibitory. Walz et al., (1990) Science, 250: 1132-1135 were able to inhibit the binding of a E-SELECTIN-lgG chimera to HL-60 cells with a monoclonal antibody directed against sLex or by glycoproteins with the sLex structure, but could not demonctr~te inhibition with CD65 or CD15 antibodies. Both groups concluded that the sLex structure is the ligand for E-SELECTIN. Patent Application No. W092/02527 assigned to the present ~c.cigne~ and incorporated herein by reference discloses and claims the foregoing minimum tetr~c;~cçh~ride structure and identi~les the groups putatively interactive with the ELAM-l receptor.
In contrast to E-SELECTIN, the ~lvpe~lies of the ligands that bind to L-SELECTIN and P-SELECTIN are not as well worked out. L-SELECTIN appears WO 94/00477 2 1 3 ~ 6 ~ 5 PCI/US93/06110 to bind a sialic acid bearing ligand based on nellr~minicl~ce treatment of peripheral lymph node high endothelial venules which inhibits L-SELECTIN recognition.
True et al., 1990, J. Cell Biol. 111, 2757-2764. Further, other studies using soluble L-SELECTIN in direct binding/inhibition assays suggests that certain carbohydrate moieties may be important ligand components including m~nnose and fucose, - particularly when sulfated or phosphorylated. Imai et al., 1990 J. Cell Biol. 111, 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 con~ sion is based on studies using antibody with this specificitythat block P-SELECTIN me~ t~d ~lh~sic)n of HL-60 cells to activated platelets orCOS cells that express P-SELECTIN. Larsen et al. (1990) Cell 63, 467-474. Other e~.;.,.çnt.c have shown that the adhesion of HL-60 cells to P-SELECTIN
transfected cells is blocked by the pent~c~rcharide isolated from milk that has the Lewis~ epitope. Recently, P-Selectin has been shown to bind to slllf~ti~es- Aruffo, A., et al. (1991) Cell, 67:35-44.
Because of the role of selectins in ~lice~ce, particularly dice~ces involving unwanted cell-cell adhesion that occurs through selectin-ligand binding on defined cell types, the ;denti~lc~tiQn and isolation of novel ligands that would permit the regula~ion of such selectin-ligand binding is sorely n~edetl WO94/004~ 35~ PCr/US93/06110 Obiects of the Invention The invention provides agonists and antagonists which bind to selectin receptors and.thus modulate the course of infl~mm~tion, cancer and related responses by mocl~ ting cell-cell adhesion events. In this aspect, the invention is directed to compounds of the formula:

QRI ~ORl ORl l~o~ \ \~ ~\ oR2 ORl X Y

(1) whelc;m each Rl is independendy H or lower aLkyl (1-4C);
R2 is H, lower aLkyl(14C), aLkylaryl or one or more additional saccharide re.ci~ e,s;
R3 is a negatively charged moiety in~luding S04--, P04--, or related group;
Y is H, oHl or lower alkyl(1-4C); and X is -CHR4(CHoRl)2CHR5ORl wherein R4 and R5 are each independently H, lower alkyl(14C), or taken together result in a five- or six-membered ring optionally co,~ g a heteroatom selected from the group concisting of O, S, and NRI;
said five- or six-membered ring optionally s1lbstit~lted with one substituent sPlecte~ from the group conci.cting of Rl, CH20Rl, ORI, OOCRI, NRI2, NHCORI, and SRl with the proviso that if X ~ Gsellts a hexose substituent R4 and R5, taken together, cannot provide a hexose sllbstitnent WO 94/00477 2 1 3 8 ~ ~ 5 PCI/US93/06tlO

In another aspect, the invention is directed to a method to synthPsi7e lactose derivatives which method comprises contacting an intenne~ t~ of the formula oR6 OR~ ~OR~<

RGO ~_ ~OR7 (2) wherein each R6 is independ~-ntly H, lower aLkyl (1-4C), or a protecting group; and whe.~ l Y' is H, OH~, OOCR6, or SR6;
wherein at least one R6, which is at the position to be s~lbstitllted~ and at most one ~dj~e.nt R6 is H and all other R6s are protecting groups; and R' is a protecting group, with an electrophile-don~ting moiety to obtain a product wherein the electrophile is sllhstit-lt~.d for the H of the OH at the position to be sl~hstit~t~i In other aspects, the invention is directed to pharm~e-lti~l compositions cont~ining the compounds of formula 1 and to methods of treating infl~mm~tion using these compocitionc~ In other aspects, the invention is directed to compounds of form~ 2 and ~d~ition~l int~rrnefli~t~s in the synthesis of selectin binding . ligands or other useful lactosyl residue-cont~ining moieties.

Modes of Carrvin~ Out the Invention It is int~de.d that all the references cited herein be inco~porated into the patent application in their entirety.
The invention provides compounds that are useful in the tre~tme~t of infl~mm~tion by virtue of their ability to bind to selectin receptors. For example, Figure 1 shows a rli~gr~mm~ti~ view of the role believed to be played by one of the selectin receptors, ELAM-1, in medi~ting infl~mm~tif~ll Blood vessels are lined with endothelial cells capable of producing the ELAM-1 surface receptor.
Lymphocytes circulating in the vessel contain on their surf~es carbohydrate s WO94/00477~3~6~1 PCr/US93/06110 ligands capable of binding to the ELAM-l 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 circllmct~n~es~ 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 infl~mm~tion. While notwishing to be limited by any particular theory, it is believed that the compounds of the present invention which bind the ELAM-l receptor, antagonize the action of the surface ligands on the circ~ ting lymphocytes and thus prevent their transfer through the blood vessel wall to cause infl~mm~tion in the surrounding tissue.
Assavs to Identify T.i~n-ls In their most general form assays for identifying lactose derivatives that act as selectin ligands involve contacting the applup,iate selectin~ L-SELECTIN, E-SELECTIN, or P-SELECTIN, with a yu~tivc; ligand and measuring its binding properties.
Several assays are available to measure the capacity of a compound to bind to L-SELECTIN, E-SELECTIN, or P-SELECTIN, and such assays are well known in the art. For example, both the selectin and the yu~tivci ligand may be in solution for a time sufficient for a complex to form consi.cting of the selectin and ligand, followed by sep~aLing the complex from uncomplexed selectin and ligand, and measuring the amount of complex formed. ~ltP.rn~tively, the amount of uncomplexed se.lectin 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 cont~rting the immobilized reagent with the non-immobilized reagent. The se11~ctin-ligand complex is separated from uncomplexed reagents, and the amount of complex formed can be dete~nin~d by m-~c11ring the amount of the non-immobilized reagent present in the complex. For example, the putative ligandcan 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 genetic~11y en~in~er cells to express high levels of L-SELECTIN, E-SELECTIN, or P-SELECTIN on their s~ ce, and wo 94/00477 ~ ~ 3 ~ 6 ~ 5 PCr/US93/06110 .

to use the cells in lieu of purified selectin Radinl~b~le~l 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 S and the number of adherent cells delc.. lh~ The number of adherent cells reflects the capacity of the ligand to bind to the selectin Represe.nt~live of the application of this type of assay is the identification of E-SELECTIN lig~nflc For eY~mple, a compl~te cDNA for the ELAM-1 receptor was obtained by PCR starting with total RNA isolated from IL-1 stimulated human umbilical vein endoth~.linm The r~sl~hing cDNA was inserted into the CDM8 plasmid (see Aruffo, A., and Seed, B., Proc. Natl. Acad. Sci. USA (1987) 84:8573) and the pl~mi~l amplified in E coli. Plasmid DNA from individual colonies was isolated and used to transfect COS cells. Positive plasmids were selecte~ by their ability to gen~o,rat~-. COS cells that support HL-60 cell adhesion. DNA sequencing positively i(lPntified one of these clones as encoding for ELAM-1 (Bevilacqua, M.P., et al., Science, (1989) 243:1160; Polte, T., et al., Nucleic Acids Res. (1990) 18:1083; Hession, C., et al., Proc. Natl. Acad. Sci. USA (1990) 87:1673). These publications are incorporated herein by reference for their disclosure of ELAM-land genetic matt-ri~l coding for its production. The complPte nucleotide sequence of the ELAM-1 cDNA and predicted amino acid sequence of the ELAM-1 protein are given in the above cited article by Bevilacqua et al., which DNA and amino acid sequences are incorporated herein by reference (see also publich~cl PCT patent application WO90/13300 which was published November 15, 1990, which is incorporated herein by reference).
A full length cDNA encoding ELAM-1 was obtained by 35 cycles of the polymerase chain reaction with 1 ,ug of total RNA t;~ d;Led from IL-1 stimulatedhuman nmbilic~l vein endothelial cells, utili7ing primers complementary to the untranslated fl~nking sequences (5'-GGTGCGGCCGCGGCCAGAGACCCGAGGAGAG-3' and 5'-GGTGTCGACCCCACCTGAGAGATCCTGTG-3'). The 2Kb insert generated was gel purified, directionally cloned into the m~mm~ n expression vector, CDM8 that had been modified by the insertion of a SalI site into the polylinker, Wo 94/00477 ~ PCr/US93/0611U

and grown in E coli (MC1061/p3). pl~cmitl.c were isolated from individual colonies and used to tr~ncf~ct COS cells. Putative E-SELECTIN encoding pl~cmi~l.c were selected based on the ability of these transfected COS cells to support HL-60 cell adh~sion 72 h posttransfection.
A positive cDNA whose sequence corresponded to the published sequence of E-SELECTIN with two nucleic acid substitutionc was used in all experim~P.nt.c.
COS cells were tr~ncf~ct~d with 1 ,ug of this pl~cmi~l DNA per 3.5 - 5.0 x 105 cells, with 400 ,ug/ml DEAE-dextran and 100 ~lM chloroquine for 4 h, followed bya brief exposure to 10% DMSO in PBS. Cells were metabolically radiolabeled overnight with carrier free 32po4 and harvested in PBS supplemPntPd with 0.02%
azide and 2 mM EDTA at 72 h posttr~ncfection for use in cell ~lhPcion studies.
E-SELECTIN transfected COS cells produced by the above method may be used to assay for glucuronyl glycolipid li~ndc, Similarly, COS cells may be tr~n.cfec~,d with cDNAs that encode L-SELECTIN and/or P-SELECTIN. The prodl~ction and char~çteri7~tion of L-SELECTIN IgG chimer~ constructs have been previously described by Watson S. R. et al., (1990) J. Cell Biol. 110: 2221-2229.
This chimera contains two complemPnt binding dom~inc, con~ci~ct~pnt with its natural expression. See Watson S. R. et al., (1991) J. Cell Biol. 115:235-243.
P-SELECTIN chimer~ was constructed in a similar manner as described by Walz, G., et al (1990) Science 250, 1132-1135, and Aruffo, A. et al.(l991) Cell, 67, 35-44, respectively. The chim~rac may be expressed in a suitable host cell, for example, 293 cells and purified. Protein A affinity chromatography is the preferred method of purific~tion. E-SELECTIN and P-SELECTIN may be constructed with llu~ ed complement binding clom~inc to standardize the size of the chimeras and to facilitate their secretion. 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 selP~tin- 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 sele~tin Wo 94/00477 2 1 3 8 64 5 PCr/us93/o6llo .

Thus, any c~ndid~t~o compound of the formula 1 may be verified to bind ELAM-l receptors by a positive result in the foregoing assays. These assays provide a simple screen for def~ g the relative effectiveness of the various members of the group con.cicting of compounds of formula 1.

Nontherapeutic Uses of ComPounds of Formula 1 In ~f~ fition to their use in treating or pr~;venf llg inflamm~tion as is further described hereinbelow, the compounds of formula 1 are useful in diagnostic and p~pd d~ory procedures both in vitro and in vivo.
Compounds of formula 1 may be conjugated to solid substrates and used for the purifi~tion of sel~ctin receptor protein from biological s~mr~les. This is conducted most conveniently by ~rranging the coupled substrate as an affinity chromatography column and applying a sample putatively cont~ining the selectin receptor protein to the affinity column under conditions wherein the selectin receptor protein is adsorbed whereas cont~min~ting m~t~.ri~lc are not. The selectin ,eceplor protein is then subsequently eluted, for example, by adjusting the eluent solution to co~ g comreting amounts of the compound of formula 1 or by adjusting pH or salt paramPt~r.c. Techniques for affinity p~ri~ tion are well understood, and routine opl;-"i,i~f;on experimPrt.c will generate the approp-iate con~ itionc for conduct of the procedure.
The compounds of formula 1 are also useful as detection reagents to determinP the presence or absence of selectin or related carbohydrate-binding receptor lig~n~.s. For use in such diagnostic assays, 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, directassay wherein the amount of complex formed is directly measured may be lltili7Pd~ltern~tively~ competition assays may be used wherein labeled selectin receptor protein is supplied along with, and in competition with, the biological sample. In some forms of the assay, it is convenient to supply the compounds of formula 1 in WO 94/00477 ?. ~'3~ ` PCr/US93/06110 labeled form so that the complex is detected dir~;lly, in ~lt~rn~te procedures, the complex may be detecPd by size sepala~ions, seeondary l~beling re~g~ntc, or other alt~rn~tce means. Suitable labels are known in the art, and include r~-lioartivelabels, fluorescent labels, enzyme labels, ehromogenic labels, or composites of these approaehes.
The compounds of formula 1 may also be used as eompetitive diagnostie reagents to detect the quantity of s~lectin receptor-binding components, such assurface lig~nds, in biological fluids. For the conduct of such assays, thè
compounds of formula 1 are labeled as deserihed above and mixed with the biological sample and cont~rt~od with the appropliate receptor protein; the iminlltiQn of binding of the labeled compound of forrnula 1 to seleetin reeeptor in the presenee of biological sample is then dett-rmin.--l The eompounds of formula 1 may also be used in im~gining studies in vivo to determine the loc~tion of seleetin lCC~plOl~i in S . For use in sueh assays, the eompounds of formula 1 are supplied with labels whieh ean be detected by in vivoim~ging teehniques, sueh as seintigraphic labels incl~ in~ indium 111, teçhn~tillm 99, iodine 131, and the like.
Teehniques for coupling compounds such as those of formula 1 to labels, chromatographic supports, or other moieties useful in employing the compounds inthe relevant procedures are well nnr~t-.rctçod in the art.
Antibodies may also be prepared to the compounds of formula 1 by coupling these eompounds to suitable earriers and admini~tering the eoupled m~tP.ri~lc to m~mm~ n or other vertebrate subjeets in standard immllni~tinn protocols with proper inclusion of adjuvants. Suitable immnnogenic carriers include, for example, Keyhole Limpet Hemocyanin (KLH), tetanus toxoid, various serum albumins such as bovine serum albumin (BSA) and certain viral proteins sueh as rotaviral VP6 protein. These coupled m~t~-ri~lc are then a(lminictered in repeated injections to subjeets such as rabbits, rats or mice and antibody titers monitored by standard immunoassay teehniques. The resulting antisera may be used per se or the antibody-secreting eells gen.-.r~t~d by the immlmi7~tion may be immortalized using standard techniques and used as a souree of monoelonal preparations which are immunoreactive with the compounds of formula 1. The WO 94/00477 ~ 1 ~ 8 6 ~ 5 PCr/US93/061 l0 .

resulting antibodies are useful in assay systems for dete~mining the presence and/or amount of the relevant formula 1 compound. Such assays are useful in monitoring the circul~ting levels of compounds of formula 1 in therapeutic tre~tm~ont.c such as those descrihed below.
S
Administration in Anti-infl~mm~torv Protocols The compounds of the invention are ~flminictered to a subject in need thereof for prophylactically plevt;~ g infl~mm~tion or relieving it after it hasbegun. "Treating" as used herein means pl~vell~ng or aml~linr~ting infl~mm?tion and/or symptoms associated with infl~mm~tion The compounds are preferably mini~tered with a ph~rm~rel-tir~lly acceptable carrier, the nature of the carrier differing with the mode of ~dmini.ctration, for example, oral ~minictration, usually using a solid carrier and I.V. ~-1minictration using a liquid salt solution carrier.
Typically, inject~hle compositionc are p-~a-cd as liquid solutions or suspensions;
solid forms sllit~hle 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. In ~dr1ition~ if desired, the vehicle maycontain minor amounts of auxiliary su-hst~nres such as wetting or emulsifying agents or pH burr~ g agents. Actual methods of p~pal.ng such dosage forms are known, or will be apparent, to those skilled in the art. See, for ~x~mple, Remin~ton's Pharm~ce~ltiral Sciences, Mack Pnhli.ching Company, Easton, PA, 17th edition, 1985. Formulations may employ a variety of excipients including, for example, pharm~ceutiç~l grades of m~nnitol, lactose, starch, m~gn~cium stearate, sodium saccharin cellulose, m~gnlocium carbonate, and the like. Oral compositions may be taken in the form of solutions, suspensions, tablets, pills,c~rsllles, sllct~in~d release ft~rmlll~tionc, or powders. Particularly useful is the a~1ministration of the subject ligand molecules directly in tr~ncderm~l formulations with permeation enhancers such as DMSO. Other topical formulations can be a~minic~red to treat dermal infl~mm~tion. In addition, transmucosal atiminictration may be effected using pen.-trantc such as bile salts or fusidic acid WO 94/00477 ~ ,6 4~ PCI/US93/06110 derivatives optionally in combination with a(ldition~l detergent moleeules. These form1]1~tionc are useful in the preparation of suppositories, for ex~mple, or nasal sprays. For suppocitoriPs, the vehicle eomposition will inelude trallition~l binders and c~rners~ sueh as polyaLkylene glyeols, or triglycçrif1Ps. Sueh suppositories may be formed from mixtures eont~ining the aetive ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%.
Tntr~n~c~l formnl~tionc will usually inelude vehieles that neither cause irrit~tion to the nasal mueosa nor ci~ni~ ntly disturb eiliary funetion. Diluents sueh as water, aqueous saline or other known snbst~n-~es ean be employed with the subjeet invention. The nasal formnl~tiQnc may also eontain preservatives sueh as, but not limited to, ehlorobutanol and bçn7~1koni1lm ehloride. A surfaetant may be present to enhanee absorption of the subjeet proteins by the nasal mueosa.
Typically, the eompositions of the instant invention will contain from less than 1% to about 95% of the active ingredient, preferably about 10% to about 50%. Preferably, between about l0 mg and 50 mg will be ~rlminicte.red to a childand between about 50 mg and 1000 mg will be ~dminictered to an adult. The frequency of a-lminictr~tion will be detP.rrninPcl by the care given based on patient responsivelless. Other err~;live doc~ges ean be readily deterrninPd by one of ordinary skill in the art through routine trials establishing dose response eurves.
In detPrrnining the dose to be ~flmini.ctered, it will be noted that it may not be decir~hlP. to eompletely bloek all seleetin reeeptors of a particular type. In order for a normal healing process to proceed, 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 oeeurring. The amount of the selPctin ligands adminictered as bloeking agents must be adjusted earefully based on the partieular needs of the patient while taking into eonsideration a variety of faetors sueh as the type ofdisease that is being treated.
The eompounds of the present invention are useful to treat a wide range of rlice~ces, for example ~nlo;~ une tlice~cP,c sueh 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 WO 94/00477 2 1 3 8 ~ ~ PCr/US93/06110 cells to acc-1m~ t~ in the tissues to the extent that they cause tissue d~m~ge, swelling, infl~mm~tion and/or pain.
Formulations of the present invention might also be ar1minictered to prevent the undesirable after effects of tissue damage rçs--lting from heart attacks. When a heart attack occurs and the patient has been revived, such as by the application of anticoagulants or thrombolytic (e.g., tPA), the endothelial lining where a clot was formed has often suffered d~m~ge When the antithrombotic has removed the clot, the l~m~gecl tissue beneath the clot and other d~maged tissue in the endothelial lining which has been deprived of oxygen become activated. The activated endothelial cells then synth~ci~e selectin receptors, for example ELAM-l receptors, within hours of the cells being ~l~m~ged. The receptors are extended into the blood vessels where they adhere to glycolipid ligand molecules on the surface of whiteblood cells. Large numbers of white blood cells are quickly captured and broughtinto the tissue surrounding the area of activated endothelial cells, resl-lting in infl~mm~tion~ swelling and necrosis which thereby decreaces the likelihood of survival of the patient.
In ~ lition to treating patients ~ur~lhlg 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 infl:lmm~tion 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. After reading the present disclosure, those skilled in the art will recognize other disease states and/or symptoms which might be treated and/or mitig~t~l by the adminictration of formulations of the present invention.

Applications of Compounds of Formula 2 The compounds of formula 2 are inter~nedi~tPs in the preparation of compounds which contain a lactosyl unit. Notably, the compounds of formula 2 W094/00477 7,~3S~ PCr/US93/06110 are useful in the pl~pa,dLion of compounds of formula 1 whose use is described hereinabove. In addition to the compounds of formula 1, ~1trrn~tive compounds cont~ining a lactose residue may also be p~ )ared, such as:
4-0-(3-0-carbonymethyl-~-D-galactopyranosyl)-3-0-[2R,S)-glyceryl]-D-glucopyranose;
4-0-(3-0-carbonymethyl-~-D-galactopyranosyl)-3-0-[2R,S)-2,3-dideoxy-2,3-difluoro-propyl]-D-glucopyranose;
4-0-[3-0-{(lR,S)-l-(carboxy)ethyl}-,13-D-gala~;Lopyl~nosyl]-3-0-[(2R,S)-glycosyl]-D-glucopyldnose;
4-0-[3-O-~(lR,S)-l-(carboxy)ethyl}-,13-D-galac~ )yl~losyl]-3-O-(a-L-fucopylanosyl)-D-glucopyranose;
4-0-[3-O-(a-Neu5Ac)-~13-D-gala.,-lopyldnosyl]-3-o-[(2R~s)-glyceryl]-D
glucopyldllose;
~0-[3-0-(oc-Neu5Ac)-,~-D-galactopyranosyl]-3-0-[(2R,S)-2,3-dideoxy-2,3-difluoro-propyl]-D-glucopyranose.

Multivalent Forms of the RecePtor Bindin~ n~s The affinity of the ligands of the invention for lccep~or can be enh~nced 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 dr~m~tirally improvesbinding to receptor. For example, Lee, R. et al., Biochem (1984) 23:4255, showedthat providing multivalent forms of lactose inhibited labeled ASOR binding to m~mm~ n hepatocytes much more effectively when the lactose was supplied as a multivalent entity; the IC50 dropped from 500 ~lM for a single valent lactose to 9 for a divalent lactosyl compound to 4 for a trivalent lactosyl compound, and with ideal or optimal spacing between the three lactose moieties to 0.007 ~lM.
The multivalency and spacing can be controlled by selection of a suitable carrier moiety. Such moieties include molecular supports which contain a mllltiplicity 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 W094/00477 213~6~7 PCr/US93/06110 of the carrier through reductive ~min~tion Reductive amination is a particularlyconvGnient way to couple aldehyde moieties to free amino groups by first formingthe Schiff base and then treating the conjugate with a reducing agent, such as ahydride reducing agent. Typically, the amino group-bearing carrier is mixed withS 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 compl~t~ the re~-~tion Particularly convenient carrier moi~ti~s to obtain multivalent forms of the invention ligands include proteins and peptides, particularly those cont~ining lysyl residues which have -amino groups available for binclinp- It is also useful to include in the peptide or protein at least one tyrosine residue, as this offers a convGnient site for l~heling~ for eY~mr1e with radioactive iodine. A particularly convGl~ient carrier to obtain a trivalent couple is the peptide Lys-Tyr-Lys.
Comp4t~ reaction of the ligands of the invention with the free amino groups on this peptide result in a trivalent moiety. Thus, compounds of the invention of the formula lOH

QRI ~oRl $EI20Rl ~o~_Nll~l~Nn~

QRI ~ORI ~2ORI ~

QRI ~ORI ~R~
'--R~I~/' wherein X, Y, and Rl, and R3 are as above defined illustrate the multivalent compounds of the invention. Of course, a variety of carriers can be used, including proteins such as BSA or HSA, a mu1tip1icity of peptides including, for e~mple, pentapeptides, decapeptides, pent~lecareptides, and the like. Preferably, 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 sulnlydlyl groups or hydr~yl groups can also be used to obtain stable link~ges For example, the carbohydrate ligands of the invention may be oxidized to contain WO 94/00477 ~)~38~64S PCI/US93/06~10 carboxyl groups at the reducing le~...i~.u~ which can then be derivaLi;~d with either free amino groups to form amides or with hydroxyl groups to form esters.

P~ ,~a~on of the ComPounds of Formula 1 The compounds of the invention of Formula 1 may be synthPci7Pcl using an intermçdi~t~ of Formula 2. The int~rmçr~i~t.o. of Forrnula 2, in one embo~lim~nt, can be prepa~ed directly from D-lactose using standard procedures. In this conversion, D-lactose is converted to the oct~cet~te in crystalline form, in over 95% yield in the method descrihed by ~u(lson, C., and Kuns, A., J Am Chem Soc (1925) 47:2052. The oct~et~t~ is, in turn, converted in more than 90% yield by the method of ~~ con, 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 con~,~,.~d by the method of Jansson, K., et al., J Or~
Chem (1988) 53.5629, in over 60% yield to the corresponding acylated trimethylsilyl ethyl lactose, which can be deprotected by deacylation in qu~ntit~tive yield to obtain 2-(trimethylsilyl)ethyl l~toci~le? 2-(trimethylsilyl)ethyl ~-D-galactopyranosyl-,B-D glucopy~ o.~i~e. ~ltern~tive protecting groups at position 1 of the ~ cch~ri~lP may also be used.

This precursor of the compounds of Formula 2 is of the formula:

OH 'OH ~OH

~0~ \Q~o~QR1 OH OH

wherein R7 is a protecting group, preferably SE or Bn, wherein SE
represents -CH2CH2SiMe3 and Bn is benzyl.
pce~tion SchPmç 1 outlines the form~tion of one embodiment of the compounds of Formula 2 from this intermediate, where Bz represents benzoyl:

WO 94/00477 2 1 3 g 64 S - Pcr/US93/06110 Reaction Sch~me 1 OH OH OH

Ho~o~oR7 OH OH
~, S~ep l . .

H3~'~ ~oR7 OH OH.
.
- 6a R7 = SE
6b R7 = ~n ~p 2 .

H~<~
OBz OBz 7a R7 = SE
7b ~7 = Bn 2 ~ 3 ~ PCr/US93/06110 In step l of the reaction scheme, the protected lactose, e.g., the trimethylsilyl ethyl derivative, is treated with an excess of 2,2-dimetho~yp,opane and dry c~mphnr 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 concentr~tPd to dryness and the base removed. In the case of benzyl lactoside, the method employed is that of D. Beith-H~1~hmi et al., Carbohydr. Res., (1967) 5:25, wherein benzyl lactoside is boiled for 3-4 hours in a large excess of dry açetQnP. cont~ining 4-toluene sulfonic acid. The reaction mixture is worked up using standard procedures to recover the product 6. This intermerli~tç is then benzoylated under suitable con~1itic)nc using, for çY~mp1P, benzoyl chloride to obtain the intermediat~
compound shown in reaction scheme as 7.
The intP.rmedi~te 7 may then be further derivatized at the free hydroxyl at the 3-position of the g1ucosidP 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 g~1~rtosyl residue is relatively unreactive. A typical scheme for utili7~tion of this key intermediate 7 is shown in Reaction .SchPme 2A. (In this scheme, Bz is benzoyl (PhCO-) and Bn is benzyl (PhCH2-).

WO 94/00477 ~ f 3 ~ 6 4 ~ ; PCr/US93/061 10 Reaction Scheme 2A

H3C O OBz OBz H3~0~0~0R7 OBz OBz CH3 / O~S~3 r ~ 0/ OBn CH3 0 OBz . ~OBz CH3><0 ~ \ \~~\ \~.~oR7 OBz 08z CH
~OBn ¦ OBn OBn 2 a R7 ~ SE
gb R7 ~ BN

OBz OBz HO~\ \~o~ \'~R7 OBz OBz C. / 0--7 ~ dBn ~ ~ a R7 = SE
OBn 10 b R7 ~ BN

(C~ J~I on ne~t page) -wo 94/00477 PCr/US93/061 10 2~38Ç~
Reaction .Sch~.me 2A

3~ (cont;--l,e~) Aco OBz 08z Ho~O~OR7 H3C~OBn t~ a ~ = SE
snO

AcO OBz OBz So3o~o~oR7 H3C~OBn 12b ~ = BN
OBn snO

OH OH OH
NaS030~,0 ~,~oR7 ~13C~OH 13 b R7 = EI(~,B) WO 94/00477 2 1 3 8 64 5 PC~/US93/06110 As shown in Reaction ,SchPme 2A, the interrnefli~te 7 is converted in two steps to intermPrli~tP 10 by tre~tmPnt under suitable conditions with protected methyl l-thio-L-fucoside. The reaction is conducted in a nonaqueous aproctic solvent in the presence of cupric bromide, tetrabutylammonium bromide and molecular sieve. (S.
Sato, et al., Carbohvdr. Res. (1986) 155:C6). The result~nt compound shown as 10- is then selectively acetylated at position 4 of D-gala~lopyldnosyl residue by the way of its 3,4- orthoester, according to literature procedure, without isolation of the int~rmedi7,te (R.U. T emiPux and H. Drigwez, J. Amer. Chem. Soc., (1975) 97:4069) to give intP.rmP~i~t~P 11. Sulfation of intermP~ tP 11 produces intP.rmedi~tP 12 which is deacylated and hydrog~Pn~tPd to yield the final product ~, a se1Pctin ligand.
In another embodiment of the instant invention, shown in reaction scheme 2B, intermediate ll may be phosphorylated to yield intermP~i~t-e 14 which upon deacylation and hydrogenation yields the final product 15. This compound would be expected to act as a selectin ligand.

WO 94/00477 PCr/US93/06110 2~3a6~ ~
Reaction .SchPme 2B

AcO OBz HO~O~OBn H3C ~OBIl 1l OBn OBn I

(Ro) ~o~o~p~oBn H3C ~OBI
OBn OBn R=C6Hs or C6HsCH2 HO OH

(NaO)2opo~ j H3C ~OH

OH

WO 94/00477 ~ 8 6 4 ~ PCr/US93/061 l0 ; .
ComPounds of the Invention and Preferred Embo~im~nt.c As used herein, alkyl (1-6C) refers to a s~t~lr~tecl straight or branched chain or cyclic hydrocarbyl residue co~ ;.l;l-g 1-6C; lower alkyl is simil~rly defined but cont~ining only 1-4C.
S As used herein, aL~cylaryl is of the formula (CH2)m-Ar wherein m is 1-10 and Ar is a mono- or bicyclic aromatic residue optionally conlS~ g one or more heteroatoms. Typical embodim~nt.c of Ar include phenyl, naphthyl, quinolyl, pyridyl, pyrimidinyl, ben7thi~7oyl, be.n7imi~7oyl, and the like.
R7 is a protecting group suitable for saccharide resid~es Typical protecting 10 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 R3 is S04-2, P04-2, or other similar charged moiety.
Additional exemplary compounds of formula 1 include those wherein X is:
6-methyl-3,4,5-trihydroxypyran-2-yl, 6-acetyl-3,4,5,trihydro,~ypyran-2-yl, 6-propylamido-3 ,4,5 ,trihydroxypyran-2-yl, 6-propylamido-2,3,4-trimethoxypyran-2-yl, 6-ethyl-2,3-dihydroxy-4-metho~yL.yl~l-2-yl~
6-N-ethylamino-2-hydroxy-3,4-ethoxypyran-2-yl, 3,4,5-tri-n-propylo~y~yl~l-2-yl, 3 ,4,5-trihydroxypyran-2-yl, 2,3 ,4-trimethoxyfuran-2-yl, 2~3-dihydroxy-4-metho~yru'all-2-yL
2-hydroxy-3,4-ethoxyfuran-2-yl, 3,4,5-tri-n-propyloxyfuran-2-yl, and 3 ,4,5-trihydroxyfuran-2-yl;
or wherein both Rs and R6 are H and all R' in X are H or methyl;
or wherein X is 2,3,4-trihydro~ylJenzoyl.
Thus, particularly preferred compounds of formula 1 are those wherein all Rl are H or methyl, Y is H, OH, OCH3 or OAc; and/or X is -CH2(CHOH)3H, 3,4,5-trihydro~yl.yl~1-2-yl, 3,4,5-trihydroxy-6-methylpyran-2-yl, 3,4,5-WO 94/00477 c PCr/US93/06~10 ~,~3~6~ ~
trimethoxypyran-2-yl, 3,4,5-trimethoxy-6-melhylpyldn-2-yl, 3,4,5-trihydroxyfuran-2-yl, 3,4,5-trimethoAyfulall-2-yl, 2,3,4-trihydroxybenzoyl, or 2,3,4-trihydroxynaphthoyl; and R3 is SO4-2, PO4-2, or other similar charged moiety.
Most preferred of the compounds of formula 1 are those wherein all R' are H, R2 is H, Y is H, ORI, or lower aL~yl.
For those compounds of formula 2 which represent intP.rmP~ tPS preferred forms are those wherein the protecting groups represented by R6 are benzyl or benzoyl, the protecting group represented by R7 is trimethylsilylethyl or benzyl, and wherein yl is H, oR6 wherein 1~6 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 g~l~ctosyl moiety. An additional preferred protecling group for positions 3 and 4 of the g~l~ctQsyl moiety is isopropylidene.
The following e~r~mpl~s are int~nflecl to ilh-str~t,- but not to limit the invention.
Example 1 Preparation of 2-(Timethvlsilyl) ethvl 2,6-di-0-benzovl-4-0-(2.6-di-0-benzoYl-3,4-0-isou,ol,ylidene-~-D-~alactopyranosyl)-~-~lucopyranoside (7a).
2-(Trimethylsilyl) ethyl 4-0-(3,4-0-isopl~,pylidene-,B-D-galactopyranosyl)-~-D-glucopyranoside (K. Jansson et al., J. Org. Chem. (1988) 53: 5629-5647; 6.6g, 13.75 mmol) was dissolved in dry pyridine (120 mL). The mixture was cooled to -45C and stirred, while benzoyl rhlori-le (9.07mL, 77.4 mMol.)was added dropwise, and stirring was contimled for 4h at -45C.
T.l.c. (8.5:1.5 toluene-ethyl acetate) revealed the presence of a major product, faster-migr~ting than the starting acetal. A small proportion of a still faster-migrating product (pent~ben70~te) was also revealed by t.l.c. The mixturewas poured into ice-water and extracted with dichlorom~th~n~ The dichlorometh~ne solution was successively washed with water, aqueous NaHCO3, and water, dried (Na2S04), and concentrated. The concentr~t~ was applied to a column of silica gel with 9:1 toluene-ethyl acetate as the eluent and gave a solid which cryst~lli7~d from m~th~nol to afford 7a (5.2g, 42.3%), [a]D +17.5 (c, 1.1,chloroform). 13C NMR (CDCl3): ~ 167.16, 166.13, 165.87, 165.83 (4xPhCO), .

111.23 ~Me2), 101.50, 100.24 (C-l, C-l'), 82.57, 77.02 (C-3', C-4), 73.65, 73.44, 73.01, 72.96, 72.06, 71.97 (C-5, C-5', C-4', C-3, C-2, C-2'), 67.21 (O_H2CH2Si),63.69, 62.72 (C-6, C-6'), 27.62, 26.28 [C~H3)2], and 17.75 (CH~CH?Si).

Example 2 Preparation of BenzYl 2.6-di-O-benzo~1-4-0-(2,6-di-O-benzoyl-3,4-O-isopro~,ylidene-~-D-~alactopyranosyl)-~-D-~lucopyranoside (7b).
A stirred and cooled (-45C, bath) solution of benzyl 4-0-(3,4-O-isoplol)ylidene-,B-D-galactopyranosyl)-~-D-glucopyranocide ( 5g, 10.6 mmol; D.
Beith-~l~hmi et al., Carbohydr Res. (1967) 5: 25) in dry pyridine (120 mL), was treated with benzoyl chloride (6 mL, 51.8 mmol), dropwise, and the stirring was contin~-ed for 4h at -45C. T.l.c. (8.5:1.5 toluene- ethyl acetate) revealed thepresence of a major product, faster-migrating than the starting acetal. A small proportion of a still faster-mi~r~ting product (pent~be.n7o~te) was also revealed by t.l.c. The ~ u-c; was poured into ice-water and t-xtracted with dichlorometh~ne The dichlorometh~nt solution was s~cceccively washed with water, aqueous NaHCO3, and water, dried (Na2SO4), and concentrated. The concentare was then applied to a column of silica gel and eluted with 9:1 toluene-ethyl acetate. On concentr~tion, the fractions corresponding to the major product gave a solid residue which crystalliæd from hot methanol to afford 7b (5.53g, 59%); m.p. 159-161C;
[OC]D -4.2 (c, 1.3, chloroform). lH NMR (CDC13): ~ 8.2-7.00 (m, 25H, arom.), 5.36 (t, lH, J 7.8 Hz, H-2'), 5.30 (dd, lH, J 8.0, and 9.5 Hz, H-2), 4.68 (d, lH, J
8.0Hz,H-1'),4.56(d, lH,J8.1Hz,H-1),3.94(dd, lH,J8.2and9.6Hz,H-3), 3.75 (dd, lH, J 8.2 and 9.7 Hz, H-4), and 1.65 and 1.35 [2s, 3H each, C(CH3)2 ];13C NMR (CDC13): ~ 167.16, 166.17, 165.90, and 165.86 (4xPHCO),111.86 (5~Me2), 102.10, 99.49 (C-l, C-l'), 82.99(C-4), 77.60 (C-3'), 74.02 (C-4'), 73.60, 73.50, 72.66 (C-2, C-2,'C-3, C-5, C-5'), 70.73 (PhCH2), 64.29 and 63.20 ( C-6, C-6'), and 28.26 and 26.88 [ (CH3)2C]; positive ion LSIMS: 889.7 (M+H)+, 781.6 M-OBn)+, negative ion LSIMS: 934.1 (M+NO2)-, 1041.1 (M+mNBA)~.

WO 94/00477 ~ . ' PCr/US93/06110 6~ --ExamPle 3 PreParation of Benzyl 2.6-di-0-benzoYl-3-0-(2,3.4-tri-0-benzvl -oc-L-fucopyranosyl)-4-0-(2,6-di-0-benzoyl-3,4-0-isoplo~ylidene-~-D-~ala-;lo~ osvl)-~-D-~lucoPv,~ oside (9b).
A ",i~u.e of compound 7b (4g, 4.5 mmol), methyl 2,3,4-tri-0-benzyl-1-thio-a-L-fucopyranoside 8 (3.6g, 7.75 mmol) and powdered 4 A molec~ r sieves (lOg) in 5:1 dichloroethane-N,N-dimethylform~mille (120 mL), protected from moisture, was stirred for 2h at room ~,llpe.~ture. Cupric bromide (2g, 9 mmol) and tetrabutylammonium bromide (0.29g,0.9 mmol) were added and the stirring was continued for 35h at room temperature. More of the donor 8 (1.2g, 2.6 mmol, in 14.4 mL of 5:1 dichloroethane-N,N-dimethylform~mi~le), cupric bromide (0.67g, 0.3 mmol), and molecular sieves 4 A (2g) were added, and the stirring was contimlt-d for 16h at room temperature. T.l.c. (9:1 toluene-ethyl acetate) then showed the presence of a major product, faster-migrating than 7b; a trace of unchanged 7b was also revealed by t.l.c. The IllL~lulc; was filtered (a bed of Celite) and the solids thoroughly washed with chlorofo"". Tne filtrate and washings werecombined and washed with aqueous NaHCO3 and water, dried and concç~
The residue was applied to a column of silica gel and eluted with 9.5:0.5 toluene-ethyl acetate. ~oncentr7~tion of the fractions corresponding to the major product fmnicht-d a solid, which cryst~lli7f d from ether to afford 9b (3.68g, 76%), based on reacted 7b. Compound 9b had m.p. 180-181C; [OC~D -8 (c, 1.1, chloroform).
'H NMR (CDC13): ~ 5.48 ( dd,l H, J 9.3 and 7.9 Hz, H-2'), 5.40 ( d, 1 H, J 3.8 Hz, H-l fuc), 5.22 ( dd, 1 H, J 8.6 and 7.3 Hz, H-2), 4.49 (d, 1 H, J 8.6 Hz, H-1), 4.42 ( d, 1 H, J 7.9 Hz, H-l'), 3.90 ( dd, 1 H, J 10.2 and 3.8 Hz, H-2 fuc), 1.49 and 1.35, ( s, 1 H each, CMe2), and 1.29 ( d, 3 H, J 6.6 Hz, H-6 fuc); l3C, (CDCl3): o 166.86-165.22 (4xPhCO), 111.44 [~(CH3)21, 100.84, 99.80 (C-l,C-1'), 63.17, 63.01 (C-6,C-6'), 28.35, 26.86 [C(~H3)2], and 17.48 (C-6"); positive ion LSIMS: 1197.9 (M-OBn)+, negative ion LSIMS: 1350.2 (M+NO2)-, 1 457.3 (M+mNBA)~.

WO 94/00477 21 386~S PCl/US93/06110 Example 4 E~cpalalion of 2-(Trimethylsilyl) ethyl 2,6-di-0-benzoyl-3-0-(2,3,4-tri-0-benzYl-a-L-fucopyranosyl)-4-0-(2,6-di-0-benzoyl-3,4-0-isop~vlidene-~-D-~alactopyranosyl)-~-D-~lucopyraoside (9a).
S A mixture of compound 7a (5.2g, 5.78 mmol), compound 8 (4.68g,10.17 mmol) and powdered 4A molecular sieves (6g), in 5:1 dichloroethane-N,N-dimethylform~mide (135 mL), protected from moisture, was stirred for 2h at room tempelatu~l;. Cupric bromide (2.6g, 11.7 mmol), and tetrabutylammonium bromide (3.77g, 11.7 mmol) were added, and the stirring was continued for a total of 48hat room tempelatulc;, additional amounts of 8 (2.34g, 5.09 mmol, in 60 mL of 5:1dichloroethane-N,N-dimethylform~mi~le), 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. (9:1 toluene- ethyl acetate) revealed the presence of a major product, faster-migrating tban 7a, Some unreacted 7a was also revealed by t.l.c. After procçccing as described for 7b (to give 9b), followed by column chromatography, compound 9a (6.7g, 88%) was obtained as an amorphous solid;
positive ion LSIMS: 1442.6 (M+Na)+, 1340.8 (M-NaSO3)+, negative LSIMS:
1396.2 (M-Na)~ .

Example 5 Preparation of Benzyl 2,6-di-0-benzoyl-3-0-(2,3,4-tri-0-benzyl-a-L-fucopyranosyl)-4-0-(2,6-di-0-benzoYl-~-D-~alactopyranosyl) -~-D-~lucoPYranoside (lOb).
Compound 9b (l.Og) in 70% aqueous acetic acid (600 mL), was stirred at 85-90C, the progress of the reaction being monitored by t.l.c.(4:1 toluene - ethyl acetate). After 2.5h, most of the starting acetal 9b was converted into a slower-migrating product. T.l.c. also in~ ated some cleavage of the a-L-fucosyl link~ge, as eviden~ed by the presence of two by-products, one of which was marginally faster-migr~ting than the product (tribenzyl fucose), and the other slower-nitrating (disaccharide product). The acetic acid was evaporated under ~liminich~d pressure ( 40C), the last traces being removed by co-evaporation with several added portions of toluene. The residue so obtained was purified in a column of silica gel wO94/0047~ 3~q~13 PCr/US93/06110 with 9:1 toluene -ethyl acetate as the eluent to give 10b (0.6g. 61.8%), as an amorphous solid. 13C NMR (CDC13): ~ 167.25, 166.80, 165.23 (4xPhCO), 100.65, 99.85 (C-l, C-l'), 98.18 (C-l fuc),79.55, 79.08 (C-3, C-4), 75.77, 73.20, 72.97,70.30 (4xPhCH2), 63.38, 62.33 (C-6, C-6'), and 17.16 (C-6 fuc); positive ion S LSIMS: 1263.7 (M+H-2H)+, 1157.7 (M-OBn)+, negative ion LSIMS: 1417.1 (M+mNBA)~, 1310.3 (M+NO2)-, 1263.2 (M-H)- .

ExamPle 6 P~ tion of 2- (Trimethylsilyl) ethYl 2~6-di-O-benzoyl-3-O-(2,3,4-tri-0-benzyl-a-L-fucoPyranosyl)-4-0-(2,6-di-0-benzoyl-~-D-~ala.;Lo~v-~losYl)-~-D-~lucopYranoside (lOa).
Compound 9a (3g, 2.3 mmol) was taken in 70% aqueous acetic acid (300 mL) and the mixture was heated, with stirring, for 2h at 85-90 (bath). T.l.c. (4:1 toluene-ethyl acetate) showed the presence of a major product with chromatographic mobility comparable to that of 10b. Processing as described for 9b (to give 10b), followed by column chromatography, gave trisaccharide diol 10a(2.3g, 79%) as an amorphous solid; [OC]D -20.6 (c, 1.1, chloroform). '3C NMR
(CDCl3): o 167.26, 166.98, 166.78, 165.04 (4 x PhCO), 101.31, 100.55 (C-l, C-l'), 98.19 (C-l fuc), 79.56, 78.98 (C-3, C-4), 75.75, 73.19, 72.98 (3 x PhCH2), 67.84(OCH2CH2Si), 63.48, 62.19 (C-6, C-6'), 18.45 (OCH2CH2Si),and 17.16 (C-6 fuc).

ExamPle 7 Preparation of Benzyl 2,6-di-0-benzoyl-4-0-(4-0-acetYl-2,6-di-0-benzoYl-,~-D-~alactopyranosvl)-3-0-(2,3.4-tri-O-benzyl-a-L-fucopyranosyl)-B-D-~lucoPyranoside (1 lb).
Compound 10b (0.56g) was dissolved in a mixture of benzene (30 mL) and triethyl orthoacetate (30 mL), cont~ining 4-toluenes~-lfonic acid (0.15g), and the mixture stirred for lh at room temperature. The acid was neutralized with a little triethylamine, and the mixture evaporated to dryness. It was then taken in 80%
aqueous acetic acid (50 mL) and stirred for 40 min at room temperature. T.l.c. (4:1 toluene-ethyl acetate) showed the presence of a major product,faster-migrating than diol 10b. The acetic acid was removed under ~liminich~l pressure, and several WO 94/00477 ~1 ~8 6~ ~ PCI/US93/06110 portion of toluene were added to, and evaporated from the residue to furnish 1 lb (0.56g, 96.6%) as an amorphous solid, [a]D -14.3 (c,l.l, chloroform). lH NMR
(CDCl3): ~ 8.20-7.00 (m, 40 H, arom.), 5.51 (t, 1 H, J 8.0 Hz, H-2'), 5.38 (d, 1 H, J 3.8 Hz, H-l fuc), 5.30 (d, 1 H, J 3.8 Hz, H-4'), 5.20 (dd, 1 H, J 8.1 and 10.0 Hz, H-2), 4.62 (d, 1 H, J 8.2 Hz, H-l), 4.44 (d, 1 H, J 7.9 Hz, H-l'), 1.82 (s, 3 H,CH3CO), and 1.34 (d, 3 H, J 6.4 Hz, H-6 fuc). 13C NMR (CDCl3): ~ 170.38 (CH3CO), 166.15, 165.72, 165.57, 164.56 (4xPhCO), 100.45, 99.23 (C-l, C-l'), 97.54 C-l fuc), 79.48 (C-3). 77.57 (C-4), 74.08, 72.94, 72.70, 70.15 (4xPhCH2), 62.54, 60.78 (C-6, C-6'), 20.59 (~H3CO), and 16.88 (C-6 fuc); positive ion LSIMS: 1307.1 (M+H)+), 1200.8 (M-OBn)+, negative ion LSIMS: 1460.9 (M+mNBA)~, 1353.6 (M+NO2)-, 1306.8 (M-H)- .

Example 8 Preparation of 2-(TrimethYlsilYl) ethvl 2.6-di-0-benzoYl-4-0-(4-0-acetyl-2,6-di-O-benzoyl-~-D-~alactopyranosyl)-3-0-(2,3,4-tri-O-benzyl-a-L-fucop~ osvl)-B-D-~alactoPYranoside (l la).
A solution of compound 10a (1.87g), in a mixture of benzene (50 mL) and triethyl ortho~cet~te (50 mL), col-l .;..;..g 4-tolu~nesulfonic acid (0.25g) was stirred for lh at room temperature. The acid was then neutralized with a few drops of triethylamine, and the mixture evaporated to dryness. The residue was mixed with80% aqueous acetic acid (100 mL) and the mixture stirred for 40 min at room tempe.~lule. Proce~cing as described for 10b ( to give 1 lb), gave the title compound lla (1.86g,89%); a white amorphous solid; [a]D -2.7 (c, 1.1, chloroform). 13C NMR (CDCl3): o 171.03 (CH3CO), 166.78, 166.35, 166.18, 165.02 (4 x PhCO), 101.27, 101.09 (C-l, C-l'), 98.17 (C-l fuc), 80.10, 78.20 (C-3, C-4), 74.67, 73.54, 73.30 (3 x PhCH2), 67.86 (O_H2CH2Si), 63.31, 61.42 (C-6, C-6'), 21.21 (~H3CO), 18.47 (OCH2_H2Si), and 17.53 (C-6 fuc); negative ion LSIMS: 1470.8 (M+mNBA)~, 1363.7 (M+NO2)-.

Wo 94/00477~S6~ PCI/US93/06110 ExamPle 9 Preparation of Benzvl 2,6-di-O-benzoyl-3-0-(2,3.4-tri-O-benzYl-a-L-fuco~y~ osvl)-4-0-(sodium 4-0-acetYl-2,6-di-0-benzoYl-~-D-~alactoPyranosYl 3-sulfate)-~-D-~lucoPvranoci~le (12b).
5A mixture of compound 1 lb (0.6g, 0.46 mmol) and sulfur trioxide-pyridine complex (0.6g, 6.3 mmol) in dry pyridinè (50 mL) was stirred for 2h at 55-60C
(bath), and then for 16h at room temperature. T.l.c. (6:1 chloroform - methanol)showed the disap~e~r~nre of llb and the presence of a single slower-migrating product. Methanol (5 mL) was added, and the mixture stirred for 15 min (to decompose excess reagent). It was then concenLr~led and purified in a column of silica gel by elution with 10:1, followed by 6:1 chloroform-m~-th~nol. On concentrAtion, the fractions corresponding to the product gave a solid residue, which was dissolved in 1:1 chloroform-methanol (30 mL) and treated with ~mberlite IR 120 (Na+) cation-exf~h~nge. resin, and the mixture stirred fo lh atroom temperature. It was then filtered and evaporated to dryness to give 12b (0.58G, 89%) as an amorphous solid; [a]D -5.1 (c, 1.8, 1:1 chloroform-m~-th~nol);
positive LSIMS: 1433 (M+Na)+, 1411.1 (M+H)+, negative LSIMS: 1563.9 (M+mNBA)-, 1386.7 (M-Na)~.

ExamPle 10 Preparation of 2-(TrimethYlsilyl) ethvl 2,6-di-O-benzovl-3-O-(2 3,4-tri-O-benzvl-oc-L-fucopyranosvl)-4-O-( sodium 4-O-acetyl-2,6-di-O-benzoyl-B-D-~alactopvranosyl 3-sulfate)-~-D-~lucoPvranoside (12a).
A mixture of compound 1 la (0.45g, 0.39 mmol ) and sulfur trioxide-pyridine complex (0.45g, 4.7 mmol) in dry pyridine (25 mL) was stirred for 2h at55-60C, and then overnight at room temperature. After proces~ing and purification, in a manner similar to the afore described, compound 12a (0.46g, 95.8%) was obtained as an amorphous solid; [CC]D ~2.2 (c,1.5, 1:1 chloroform-meth~nol); positive ion LSIMS: 1442.6 (M+Na)+, 1341.1 (M-NaSO3)~, negative ion LSIMS: 1395.5 (M-Na)~.

WO94/00477 ~ 645 PCr/US93/06110 Example 11 u~dLion of O-a-L-fucou~lanosyl-(1~3)-O-rsodium ~-D-~alactop~ranos~ll 3-sulfate-(1- ~4)l-D-~lucoPYranose (13b).
Compound 12b (0.58g) in m~th~nol (50 mL), conl;.;..il-g a catalytic amount of sodium msthoxide~ was stirred overnight at 45-50. T.l.c. (13:6:1 chloroform-mPth~nol-water) showed the presence of a single slower-migrating product. After cooling to room tem~e~Lulc;, Amberlite IR 120 (H+) cation-exrh~nge resin was added till the llli~lulc became neutral (pH paper). It was then filtered directly into a flask co~ g Ambçrlite IR 120 (Na+) cation-exchange resin, and the mixture stirred for 45 min. It was then conr,entrflt-d and the residue repeatedly extracted with hexane-ether mixture to remove medlyl ben7O~t~ The partially-protected intermedi~te so obtained (0.38g,) was sufficiendy pure to be utilized directly in the next step; negative ion LSIMS: 928.1 (M-Na)~. A portion (0.35g), without furtherpnrifir,atiQn, was taken in 80% aqueous msth~nol (30 mL), cont~ining 10%
p~ (1illm-on-carbon 0.35g). The mixture was stirred overnight at room temperature under a slight overpressure of H2, when t.l.c. (5:4:1, or 13:6:1 chloroform-mcth~nQl-water) in(lir~ted the presence of a slower-migr~ting product, together with traces of some faster-migr~ting cont~min~ntc ( presumably due to incomplete hydrogenolysis). The mixture was filtered ( Celite bed) directly onto Amberlite IR
120 (Na+) cation-exch~nge resin, and the solids thoroughly washed with aqueous methanol. After stirring with the resin for lh, the ~ urt; was filtered and conePntr~ted to a small volume, which was applied to a column of silica gel and eluted with 5:4:1 chloroform-meth~nQ1-water. Fractions corresponding to the product were pooled, concentrated to a small volume and treated with Amberlite IR
120 (Na+) cation-exchange resin. The resin was filtered off and washed with water, and the filtrate and washings combined, refiltered ( 0.2 ~lM CellnlQse acetate syringe filter), and lyophilized to give 13b, (183 mg, 84.3%; [OC]D -20.5 (c, 0.6, water). 1 H NMR (D20): ~ 5.45 [d, 1 H, J 4.13 Hz, H-l fuc (~)], 5.39 [d, 1 H, J
3.81 Hz, H-l fuc (oc)], 5.18 (d, 1 H, J 3.81 Hz, H-l), 4.66 (d, 1 H, J 7.93 Hz, H-1'), 4.55 [d, 1 H, J 7.62 Hz, H-l (,~)]; negative ion LSIMS: 567.5 (M-Na)~, 421 (M-Na-Fuc)~.

-wo94'4"2~3a64~j PCr/US93/06110 Example 12 Fre~dLion of 2-(TrimethylsilYl ethyl O-a-L-fucopvranosyl-(1~3)-O -r sodium 13-D-~ala~;~o~ osYl 3-sulfate-(1~4)1-~-D-~lucopyranoside (13a).
Compound 12a (0.45g) was O-deacylated in methanolic sodium mrthQxide (50 mL), exactly as described for 10, to afford the corresponding partially benzylated interme~ te (0.29g), which showed positive ion LSIMS: 983.9 (M+Na)+, 882.1 (M-NaSO3 ), negative ion LSIMS: 938.0(M-Na)~ . This compound (0.24g) without any further pmifie~tion, was subjected to catalytic hydrogenolysis in 80% aqueous meth~nQl (30 mL) in the presence of 10% p~ lm-on-carbon (0.24g), and then processed in a manner analogous to the afore described to afford compound 13a (125 mg, 72.7%), as a white fluffy m~teri~l; [a]D -49.2 (c,0.6. water).lH NMR
(D2O): ~ S.45 (d, 1 H, J 4.22 Hz, H-1 fuc), 4.55 (d,1 H, J 8.06 Hz, H-1'),4.49 (d, 1 H, J 8,44 Hz,H-1), 4.32 (dd, 1 H, J 3.45 and 9.98 Hz, H-3'); positive ion LSIMS:713.8 (M+Na)+, negative ion LSIMS: 667.6 (M-Na)~ .
Example 13 Preparation of a Multivalent T i~n(l N,6N,6N' Tris (20) LYs-TYr-L~s Compound 13a or 13b, prepared in Example 127 may be derivatized to the peptide Lys-Tyr-Lys to obtain the trivalent conjugate derivatized at the two -amino lysine groups and the a-amino N-sormin~l of the peptide. To obtain this trivalent compound, 50 111 of 2 mM peptide Lys-Tyr-Lys (100 nmol) in 100 mM
sodium carbonate, pH 9, are placed in a small Eppendorf tube cont~ining 5 ~11 of200 mM 20 (1 mmol), and the sample is evaporated to dryness in a SpeedVac for about 30 min~lt~s After evaporation, 50 111 of 800 mM NaCN BH3 (recrystallized, 40 ~lmol) in 100 mM sodium carbonate, pH 9, is added and the mixture is incubated for 48 hours at 55C. The re.s-llting incubated mixture is run on a GPC peptide HPLC
sizing column and fractions are collected and assayed for protein content by BCAprotein assay. Protein-cont~ining fractions are pooled, lyophilized and submitted for mass spectroscopy.
The results would show the formation of the derivatized peptide as co~t~ining 1, 2 or 3 moieties of compound 13a or 13b.

WO 94/00477 kl 3 86 ~ 5 PCr/US93/06110 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.

Selectin Li~and ~ru~)c;lLies of 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 glycoIipid, 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 con~i~;..;--g imM Ca. While the plate is being blocked, biotin labelled goat F(ab')2 IgG (Fc specific) and streptavidin-alkaline phosphatase diluted 1:1500 in 1 % BSA-PBS (lmM Ca) are combined with either the E- or L-Selectin-IgG chimera (L91-10) at 200 ng/mL and incubated at 37 C for 15 mimltes to allow a complex to form. This provides a soluble "multivalent" receptor. Compounds 13a and 13b were added at final con~entr~tio~c ranging from 1.5 to 5.0 mM to thesoluble 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 mim tPs 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" ,t;ceplor reacted with only the ligand evaporated to the microtiter well. This was considered "100 % bin~lin~: " 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.
It is appa~ t from Table 1 that both compounds 13a and 13b inhibit binding of E selectin to 2,3 sLex glycolipid. Over the three concentrations tested 13b was the better inhibitor with the greatest difference apparent at 5mM
conrentratiQn. At this concentration 13b showed 82.5% inhibition compared to 48% for 13a.

WO 94/00477 PCr/US93/06110 ~6~ ~
2 Table 1 INHIBlTION OF E-SELECTIN BINDING TO sLeX

COMPOUND CONC. (mM) % INHIBITION
s 13a 1.25 30 2.50 34 5.00 48 13b 1.25 48.5 2.50 45.4 5.00 82.5 It is appalt;nt from Table 2 that both compounds 13a and 13b also inhibit binding of L selectin to 2,3 sLex glycolipid. However, the dirre,ence here was considerably greater than the difference in % inhibitinn for binding to E selectin. For example, at 1.25 mM, 13b surprisingly showed 90% inhibition. 100% inhibition wasobserved at 2 mM and S mM. In m~rk.o.~l contrast, 13b displayed only 13% inhihition at 1.25 mM and a maximum inhibition of 47% at 5 mM.

Table 2 INHIBITION OF L-SELECTIN TO sLex CONC. (mM) % INHIBITION
13a 1.25 13 2.50 27 5.00 47 13b 1.25 90 2.50 100 5.00 100

Claims (12)

Claims What is claimed is:

1. A compound of the formula:

wherein each R1 is independently H or lower alkyl (1-4C);
R2 is H, lower alkyl(1-4C), alkylaryl or one or more additional saccharide residues;
R3 is a negatively charged moiety including SO4--, PO4--;
Y is H, OH or lower alkyl(1-4C); and X is -CHR4(CHOR1)2CHR5OR1 wherein R4 and R5 are each independently H, lower allyl(1-4C), or taken together result in a five- or six-membered ring optionally containing a heteroatom selected from the group consisting of O, S, and NR1;
said five- or six-membered ring optionally substituted with one substituent selected from the group consisting of R1, CH20R1, OR1, OOCR1, NR12, NHCOR1, and SR1 with the proviso that if X represents a hexose substituent R4 and R5, taken together, cannot provide a hexose substituent.

2. The compound of claim 1 wherein all R1 are H.

3. The compound of claim 1 wherein R2 is H.

4. The compound of claim 1 wherein Y is H or OH.

5. The compound of claim 1 wherein X is -CH2(CHOH)3H, 2,3,4-trihydroxybenzoyl, or is a 3,4,5-trihydroxy or 3,4,5-trimethoxypyran-2-yl or furan-2-yl.

6. The compound of claim 1 wherein one of R4 and R5 is H and the other is H, lower alkyl (1-4C), or phenyl.

7. The compound of claim 6 wherein said alkyl is methyl.

8. The compound of claim 6 wherein both R4 and R5 are H.

9. The compound of claim 1 wherein R4 and R5 taken together are 3,4,5-trihydroxy or 3,4,5-trimethoxypyran-2-yl or furan-2-yl.

10. The compound of claim 1 wherein all R1 are H, R2 is H, R3 comprises S04--and X is a fucosyl residue.

11. A method to synthesize lactose derivatives, said method comprising contacting a compound of the formula:
wherein each R6 is independently H, lower alkyl (1-4C), or a protecting group;
wherein Y1 is H, OH, OR7OOCR7, or SR7;
wherein at least one R6 which is at the position to be substituted, and at most one adjacent R6 is H and all other R6s are protecting groups;
wherein R7 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.

12. The method of claim 11 wherein the compound of said formula is selected from the group consisting of:
benzyl 6-O-benzoyl-3-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-4-O-(6-O-benzoyl-.beta.-D-galactopyranosyl)-.beta.-D-glucopyranoside;
benzyl 6-O-benzoyl-3-O-(2,3,4-tri-O-benzyl-d-L-fucopyranosyl)-4-O-(6-O-benzoyl-3,4-O-isopropylidene .beta.-D-galactopyranosyl)-.beta.-D-glucopyranoside;
benzyl 3-O-(2,3,-tri-O-benzyl-.alpha.-L-fucopyranosyl)-4-O-(3,4-O-isopropylidene-.beta.-D-galactopyranosyl)-.beta.-D-glucopyranoside;
benzyl 2,6-di-O-benzoyl-3-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-4-O-(2,6-di-O-benzoyl-3,4-0-isorpopylidene-.beta.-D-galactopyranosyl)-.beta.-D-glucopyranoside;
benzyl 2,6-di-O-benzoyl-4-O-(2,6-di-O-benzoyl-3,4-O-isopropylidene-.beta.-D-galactopyranosyl)-.beta.-D-glucopyranoside;
2-(Trimethylsilyl) ethyl 3-O-(2,3,4-tri-O-benzyl-L-fucopyranosyl)-4-O-(2,6-di-O-benzoyl-.beta.-D-galactopyranosyl)-2,6-di-O-benzoyl-.beta.-D-glucopyranoside; and benzylO-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-(1-3-[O-(2,6-di-O-benzoyl-3,4-O-isopropylidene-.beta.-D-galactopyranosyl)-(1-4)]-2,6-di-O-benzoyl-.beta.-D-glucopyranoside.