CA2164961A1 - Fucosylated glycosides as inhibitors of bacterial adherence - Google Patents

Abstract

Mono-, di-, tri- or oligosaccharide glycoside derivatives having at least one terminal group which is derived from L-fucose. The compounds are useful for therapy or prophylaxis in conditions involving infection by Heliobacter pylori of human gastric mucosa. Another object of the present invention is to provide a process for their preparation and pharmaceutical compositions.

Description

FUCO8YI~TED GLYC08I~5R A8 IN}IIBITO}?.8 OF BACTERIl~I, AnRF~ R

FlELD OF THE INVENTION

The present invention relates to the use of 1-fucose-containing glycoside derivatives for the preparation of pharmaceutical compositions for the treatment or prophylaxis of conditions involving gastrointestinal infections by Helicobacter pylori, a method of treating such conditions using the derivatives, as well as novel glycoside derivatives.

BACKGRoUND OF THE INVENTION

Helicobacter pylori is a microaerophilic spiral shaped organism (originally assigned to the genus Campylobacter) which is found in the stomach and generally appears to have an exclusive habitat in the human gastric mucosa. It has been estimated that this bac~erium infects the gastric mucosa of more than 60% of adult humans by the time they are 60 years old. Moreover, H.
20 pyl ori has been implicated as a contributing factor in a number of pathological conditions, including acute (type B) gastritis, gastric and duodenal ulcers, atrophic gastritis, and gastric adenocarc:inoma.

Tissue tropism of bacteria is partly governed by the ability of a bacterial strain to adjust to the local chem ical environment in its specific habitat. In addition,adherence is a nec~ss~y prerequisite for colonization in order to prevent removal from the new habitat, e.g. through peristalsis in the gastrointestinal tract. In mammals, bacteria adhere to proteins or glycoconjugates (glycosphingolipids, glycoproteins) on or in the vicinity of epithelial cell surfaces (mucus), and a number of specific bacterial adhesin-protein and adhesin-carbohydrate interactions have been described in the ,35 literature.

With respect to H. pylori, studies in model systems such as mouse adrenal Y-l cells (see D. G. Evans, D. J., Jr. Evans, and SUB~ JTE SHEEr wo gs/~527 ~ 9 6 ~ PCT/SE94/00604 D. Y. Graham, (1989) Infect. Immun. 57, 2272-2278) have suggested that surface-associated flexible fibrillar structures that surround this bacterium function as adhesins or colonization factor antigens to mediate the binding of H.
5 pyl ori to cellular sialic acid-containing glycoprotein receptors.

SUMMARY OF THE INVENTION

The invention concerns the use of mono-, di-, tri- or oligosaccharide glycoside derivatives having at least one terminal group Y, as defined below, derived from L-fucose, said derivatives being compounds of the general formula Ia, Ib, Ic, Id, Ie or If Ia Ib Ic A--Z5--B--Z6-c--z7--R A-Z8-B-Zg-C-zlo~D-zll--R

Id Ie A--Zl2--B--Zl3-C-Zl4-D-Zls~E~Zl6~R

wherein Zl, Z2~ Z3~ Z4~ Z5~ Z6~ Z7~ Zg~ Zg~ Zlo, Zll~ Zl2~ Zl3~ Zl4~
Z15 and Z16 independently are O, S, CH2, or NR25, where R2s is hydrogen, Cl_24-alkyl, C2_24-alkenyl, C1_24-alkylcarbonyl, or benzoyl optionally substituted with hydroxy, amino, Cl_4-alkyl, C1_4-alkoxy, nitro, halogen, phenoxy, or mono-or di-halogen-Cl_4-alkyl;

SUBs ~ JTE SHEET

wo 95/00527 2 1 6 ~ 9 6 1 PCT/æ94/00604 y ~ C~,~ A i~

R" ~ R-C
B i9 R3~ ; C 19 ~ ;

R2~ R~- RX R1C
10 R'~ ~ E i8 '~ ~_ ~ R~

15wherein the wavy line in Y, A, B, C, D and E signifies a bond which is either in the ~- or in the ~-configuration;

~Rl, R2, and R3 each in~ep~n~ntly are H, halogen, ~zido, guanidinyl, br~h~ or unbrAn~he~ Cl_24-alkyl, c2_2g-alkenyl, c2_24-alkynyl, c3-8-CYClalkYl ~
3-8 cycloalkyl-Cl_24-alkyl, or Cl_l2-alkoxy-Cl_l2-alkyl group which is optionally substituted with hydroxy, amino, halogen, or oxo; aryl or aryl-Cl_4-alkyl optionally substituted in the aryl moiety with hydroxy, amino, Cl_4-alkyl, Cl_4-alkoxy, nitro, halogen, phenoxy, or mono- or di-halogen-Cl_4-alkyl;
tri(Cl_4-alkyl)silylethyl; oxo;
a ~LoU~ =CR4Rs wherein R4 and R5 independently are H, or cl_4-alkYl;
or a ~l~U~ XR10 wherein X is O, S, NR20, or =N-, and Rlo ~s H, branched or unbranched Cl_24-alkyl, C2_24-alkenyl, C2-24-alkYnYl, C3-8-cycloalkyl, 3-8 cycloalkyl-Cl_24-alkyl, or Cl_l2-alkoxy-C1 12-alkyl group which is optionally substituted with hydroxy, amino, halogen, or oxo; aryl, aryl-Cl_4-alkyl, or heterocyclyl-Cl_4-alkyl optionally substituted in the aryl or heterocyclyl moiety with hydroxy, amino, SUB~ JTE SHEE~T

WOg5/~7 ~6 49~ ~ PCT/SE94100604 C1_4-alkyl, Cl_4-alkoxy, nitro, halogen, phenoxy, or mono- or di-halogen-C1_4-alkyl; tri (Cl_4 alkyl)silylethyl; tri(C1_4-alkyl)silyl;
tri(C1_4-alkyl)silylethoxymethyl; the acyl residue of a naturally occurring amino acid; C1_24-alkylcarbonyl;
C2_24-alkenylcarbonyl;
C3_8-cycloalkyl-C1_24-alkylcarbonyl; arylcarbonyl; or terpenyl; and R20 is H, C1_24-alkyl~ C2_24-alkenyl, C1_24-alkylcarbonyl, or benzoyl or phthaloyl optionally substituted in the benzene ring with hydroxy, amino, Cl_4-alkyl, Cl_4-alkoxy, nitro, halogen, phenoxy, or mono- or di-halogen-C1_4-alkyl;

1A ~ R2A~ R3A~ R4A~ Rls~ R2s~ R3B~ R4B~ Rlc~ R2c~ R3c~ R4c~
R1D~ R2Dr R3D~ R4D~ R1E~ R2E~ R3E~ and R4E each in~eren~ently is as defined for Rl, R2, and R3 above, or is a group of the formula VII

YZl VII

wherein Y and Zl are as defined above;
with the provisos that one of R1B~ R2B~ R3B~ or R4B is Z3, Z5, Z8 or Z12~
~hat one of R1c~ R2C~ R3C, or R4c is Z6~ Z9 or Z13, that one of R1D~ R2Dr R3D~ or R4D is ZlO~ or Z14, that one of R1E~ R2E' R3E' or R4E is Z15, that at least one and at the most five of R1A~ R2A~ R3A~ R4A~ R1B' R2B~ R3B~ R4B~
R1C~ R2C~ R3C~ R4c~ R1D~ R2D~ R3D~ R4D~ R1E~ R2E~ R3E~ and 3 R4E is a group of the formula VII, and that the configurations of the substituents R1A~ R2A' R3A~ and R4ACH2 in A, the configurations of the substituents R18, R2B, R38, and R48CH2 in B, the configurations of the substituents R1C, R2C' R3c~ and R4CCH2 in C, the configurations of the substituents R1D ~ R2D' R3Dr and R4DCH2 in D, and the configurations of the substituents R1E, R2E' R3E' and R4ECH2 in E
independently are D-gluco, L-gluco, D-galacto, SUB~ ~ .JTE SHEET

-W095l~s27 21 6 ~ 9 61 PCT/SE94/00604 L-galacto, D-manno, L-manno, D-talo, L-talo, D-allo, L-allo, D-altro, L-altro, D-gulo, L-gulo, D-ido, or L-ido;

~ 5 is a branched or unbranched C1_24-alkyl, C2_24-alkenyl, c2-24-alkynyl, C3-8-cycloalkyl, C3_8-cycloalkyl-Cl_24-alkYl, Cl-l2-alkXY-Cl-l2-alkYl, Cl_24-alkylcarbonyl, C2_24-alkenylcar~onyl, or C3_8-cycloalkyl-Cl_24-alkylcarbonyl group which is optionally substituted with hydroxy, amino, halogen, or oxo; an aryl, aryl-Cl_4-alkyl, arylcarbonyl or aryl-Cl_4-alkylcarbonyl group optionally substituted in the aryl moiety with hydroxy, amino, C1_4-alkyl, Cl_4-alkoxy, nitro, halogen, phenoxy, or mono- or di-halogen-Cl_4-alkyl; terpenyl;
tri(Cl_4-alkyl)silylethyl; heterocyclyl;
e t heterocyclyl-C1_4-alkyl; or -~ heterocyclyl-C1_4-alkylcarbonyl;

a group o~ the formula II or IIa R3o~(CH2)q~StO)m-CH2CH2- II
tR3O~(CH2)q~S(O)m~CH2~2CH~CH2~IIa wherein R30 is H, carboxy, C1_4-alkoxycarbonyl, hy~-o~, amino, or a matrix MA, q is an integer from 1 to 24, and m is O or 2; or a group of the formula III or IIIa Phe-s(o)m-cH2cH2- III
tPhe-S(O)m-CH2]2CH~CH2~ IIIa wherein m is as defined above, and each Phe is 35 phenyl optionally substituted with hydroxy, amino, Cl_4-alkyl, Cl_4-alkoxy, nitro, halogen, phenoxy or mono- or di-halogen Cl_4-alkyl; or phenyl-C1_4-alkyl optionally monosubstituted in the phenyl moiety SUB~ JTE SHEET

W095/00S27 2 ~ 6 4 9 6 ~ PCT/SE94100604 with hydroxy, amino, C1_4-alkyl, Cl_4-alkoxy, nitro, halogen, phenoxy, or mono- or di-halogen-C1_4-alkyl;

a group of the formula IV

R40cH2cH(cH2Rso)cH2- IV

wherein R40 and R50 independently are halogen; or0 a group Q-(Spacer) r~, where r is an integer O or l, and Q is a matrix MA or a group -COO-MA;

in therapy, especially for the treatment or prophylaxis in humans of conditions involving infection by Helicobacter pylori of human gastric mucosa. Another aspect of the invention relates to the use of said compounds for the preparation of pharmaceutical compositions for use against the above mentioned conditions.

DETAILED DESCRIPTION OF THE INVENTION

In the present context, the terms "Cl_4-alkyl", "C1_8-alkyl" and "Cl_24-alkyl" as a separate group or as part of a group designates alkyl ~LOu~ with 1-4, 1-8 or 1-24 carbon atoms which may be straight or branched such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert.butyl, dimethylbutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, hexadecyl, octadecyl, etc.
In the carbon chain the definition "Cl_24-alkyl" is used herein, but also shorter number of carbon atoms in the carbon chain is possible as "Cl_8-alkyl" or "Cl_4-alkyl".
The term "Cl_4-alkyl" is used herein when substituents are defined.
The term "C3_8-cycloalkyl" as a group or as part of a group designates a cyclic alkyl group with 3-8 carbon atoms such as SUB~ JTE SHEET

WOg5/~7 21 6 ~ ~ ~1 PCT/SE94/00604 _7_ cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl.
The term "C2_24-alkenyl" designates unsaturated alkyl groups with 2-24 carbon atoms which may be straight or branched, preferably straight, in which the double bond may be present anywhere in the chain, for example vinyl, l-propenyl,

2-propenyl, hexenyl, decenyl, hexadecenyl, octadecenyl. The term "C2_24-alkynyl" designates an alkyl group with 2-24 carbon atoms and incorporating a triple bond, e.g. ethynyl, 1-p~o~yl.yl, 2-propynyl, 2-butynyl etc. The term "halogen"
designates Cl, Br, I and F, preferably F and Cl.
The terms "C1_4-alkoxy" and "Cl_24-alkoxy" designate yLou~
comprising an oxa function substituted with an alkyl group as defined above.
The terms "aryl" and "aryloxy", either as a separate group or as part of a group, designates phenyl or naphthyl, preferably phenyl.

The term "aryl-amide" defines either aryl-NH-C(O)- e.g.
anilids, or aryl-C(O)-NH- e.g.benzamide.

The term ~terpenyl moiety" designates ~G~ derived from some of the various unsaturated hydrocarbon compounds generically known as the terpenes, namely the monoterpenes and the sesquiterpenes, as well as hydroxy or oxo derivatives thereof.
Examples of such ~.ou~ are myrcenyl, (-)-limonenyl, terpineloyl, (+)-~-pinenyl, geraniolyl, (-)-mentholyl, (-)-camphoryl, farnesolyl, ~-eudesmolyl, and manoolyl.
In the present context, the term "oligosaccharide" designates an oligosaccharide containing 4-10 monosaccharide units, preferably 4-7 monosaccharide units, the monosaccharide units being selected from aldohexo-ec (i.e. D-glucose, L-glucose, D-galactose, L-galactose, D-mannose, L-mannose, D-talose, L-talose, D-allose, L-allose, D-altrose, L-altrose, D-gulose, L-gulose, D-idose, or L-idose) or their derivatives, where the oligosaccharide may be linear or branched with the proviso that SlJB~ I 11 ~E SHEEr woss/~7 PCT/SE94/006W
2I'S~'9~ 8-there are no more than sëven monosaccharide units in the longest chain in the oligosaccharide.

As indicated above, the wavy lines on the carbon atoms S neighbouring the ring oxygen atoms in groups Y, A, B, C, D, and E signify that the bonds in question which are glycosidic bonds have either the ~- or the ~-configuration. It is clear that each of the bonds in question on a particular group Y, A, B, C, D, and E may assume the ~- or the ~-configuration independent of the corresponding bonds on the other groups.

A mono- or di-halogen-Cl_4-alkyl group may be substituted in any position and if substituted with 2 halogen atoms, the halogen atoms may be the same or different.
The term ~'heterocyclyl~' designates a monocyclic 5- or 6-membered, or a fused bicyclic (each ring being 5- or 6-membered), aromatic or partly or fully saturated heterocyclic group containing from one to four hetero atoms per ring, the heteroatoms being selected independently from O, S and N and bound either via a carbon atom or v~a a nitrogen atom. Typical but non-limiting examples of such groups may comprise pyrrolyl, pyrazolyl, pyridinyl, thienyl, thiazolyl, oxazolyl, imidazolyl, isoxazolyl, isothiazolyl, furyl, pyrazinyl, pyrimidinyl, pyridazinyl, 2H-1,3-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 2H-1,3-thiazinyl, 4H-1,3-thiazinyl, 6H-1,3-thiazinyl, lH-1,2,3-triazolyl, 2H-1,2,3-triazolyl, lH-1,2,4-triazolyl, 4H-1,2,4-triazolyl, indolyl, purinyl, piperidyl or piperidino, morpholinyl or morpholino, piperazinyl, tetrahydrofuryl, thiazolidinyl, oxazolidinyl, imidazolidinyl, isoxazolidinyl, isothiazolidinyl, pyrrolidinyl, lH-tetrazolyl, or 2H-tetrazolyl.

The term "acyl residue of a naturally occurring amino acid"
designates the acyl residue of the L-amino acids occurring in proteins in nature, e.g. alanyl, valyl, leucyl, isoleucyl, prolinyl, phenylalanyl, tryptophanyl, methionyl, glycyl, seryl, threonyl, cysteinyl, ~yLG~yl, asparagyl, glutamyl, lysyl, SUB~ 111 IJTE SHEET

W095/~ ~ 6~g~6~ PCT/SE94/00604 _g_ arginyl, histidyl and the acyl residues of aspartic acid and glutamic acid, the acyl residue referring both to the carboxy group next to the amino function as well as the carboxy group at the end of the respective side chains, preferably, however, the carboxy ~lo~ next to the amino functions.

The term "sphingoid" refers to D-erythro-2-amino-1,3-octadecanediol, its homologs and stereoisomers and to hydroxy and unsaturated derivatives thereof, including ceramide (see further definitions in Journ. of Lipid Research, vol. 19, (1978), 617-631).

The term ~steroid~ refers to well-known steroids as cholesterol, cortisone, hydrocortisone, corticosterone, betamet~asone, prednosolone, prednisone etc.

The term "matrix" as used herein and designated as "MA"
signifies any organic or inorganic, polymeric or macromolecular structure to which the aglycon part of the O-, S-, C-, or N-glycosidic compound of the formula Ia, Ib, Ic, Id, Ie or If is attached either covalently or by e.g. hydrophobic interaction. Examples of such matrices are residues of proteins, glyco~G~eins, polypeptides, polysaccharides, liposomes, emulsions, plastic polymers and inorganic materials.
Residues of proteins are preferably bonded through nucleophilic y~OU~S in the proteins, e.g. groups such as amino, hydroxyl and mercapto groups. Proteins or polypeptides themselves may be any of a wide variety of substances, in particular biologically compatible proteins such as globulins, albumins such as human serum a~bumin (HSA), bovine serum albumin (BSA) or sheep serum albumin (SSA), ovalbumin, fibrins, or "key-hole" limpet haemocyanin (KLH), glycoproteins such as bovine or human whole casein or lectins, and the like. Other examples of such matricee~ are synthetic polymers where one or several amino acids are coupled to a polymer of defined size(s), e.g.
polylysi.ne or oligolysine. In the various proteins or polypeptides, the linkage to the remainder of the group R may be through amino groups or through carboxyl groups.

SUB~ JTE SHEEr woss/~s27 2 1 6 ~9 `~ 1 PCT/SE94100604 The polysaccharides, to which the o-, S-, C-, or N-glycosidic compounds ~re attached, may be any of a wide variety of polysaccharides. The aglycon part of the compound of formula Ia, Ib, Ic, Id, Ie or If may be bonded through hydroxyl groups on ordinary polysaccharides such as cellulose, sepharose, starch or glycogen, through amino groups on amino saccharides such as chitosane or aminated sepharose, and through mercapto gLou~s of thio-modified polysaccharides.

Liposomes may be any biocompatible, biodegradable microesicular system compose of one or several bilayers surrounding aqueous compartments, within which a variety of agents can be encapsulated: hydrophobic agents in the lipid bilayers and hydrophilic agents in the inner aqueous space. The lS physicochemical properties of the liposomes are mainly dependent on the lipid composition.

L~posomes are composed of phospholipids, such as egg yolk phospholipids, soya phospholipids, synthetic phosphatidylcholine e.g dimyritoylphosphatidylcholine (DMPC) and/or dipalmtoylphosphartidylchlorine (DPPC) or purified phosphatidylcholines of vegetable origin or other lipids, such as galactolipids, sphingolipids or glycosphingolipids.

Emulsions are heterogenous mix~u~e~ of two or more imiscible liquids. To stabilize these systems an emulsifier is added. The emulsifier is oriented at the interface of the imisible liquids and usually only one phase persist in dropted form.

Emulsions fall into two general categories. The heterogenous system described by droplets of an organic liquid dispersed in a continuous water phase is called oil-in-water emulsion to/w).
Alternatively, the heterogenous system described by droplets of water dispersed in a continuous oil phase is called water-in-oil emulsion (w/o).

Any vegetable oil such as soybean oil, safflower oil, sesameoil, peanut oil, cottonseed oil, borago oil, sunflower oil, SUB~ ITE SHEET

wo 95/00527 2 1 6 ~ 9 61 PCT/SE94/00604 corn oil, olive oil, medium chain triglycerides (such as Miglyol R ), or acetylated monoglycerides may be used as internal or continuous phase.
Examples of plastics to which the aglycon part of the compounds of the formula Ia, Ib, Ic, Id, Ie or If may be attached are aminated latex, thiolated, aminated, or hydroxylated polys~y~ene, polyacrylamide and polyvinyl alcohol. Other possible carriers are beads and gels of carbohydrate origin or polymers where carbohydrates are used in combination with other polymeric materials such as sephacryl. These gels are further substituted with ~,ou~ such as amino, thiols, cyano, active esters and disulfides. The plastics in question may be in the form of e.g. beads or film.

lS Examples of inorganic material, to which the aglycon part of the compounds of the formula Ia, Ib, Ic, Id, Ie or If may be attached are silicon oxide materials such as silica gel, zeolite, diatomaceous earth, or the surface of various glass or silica gel types such as thiolated or aminated glass, where the silica gel or the glass may be in the form of e.g. beads.
Another example of an inorganic material is aluminium oxide.

Particularly preferred matrix MA is human serum albumin (HSA), bovine serum albumin (BSA) and polyacrylamide (PAA).
An interesting embodiment of the invention i8 when the compound of formula Ia, Ib, Ic, Id, Ie or If comprises a matrix MA, said matrix incorporating a multiplicity (i.e. 2 or more, such as 2-lOO when the matrix is a protein such as BSA or HSA, or lO-lO,OOO when the matrix is a polymer such as polyacrylamide) of moieties of the formula Ia, Ib, Ic, Id, Ie and If. It is contemplated that the presence of several such moieties will substantially enhance the inhibiting effect of the entire compound due to a multivalency-effect thereof on the bacteria.
It is also possible that the presence of several moieties of the formula Ia, Ib, Ic, Id, Ie and If may even lead to agglutination of the bacteria.

SUB~ ~ JTE SHEEr wo gs/00s27 2`1~6 ~ 6 ~ PCT/SE94/00604~

When, in connection with the definition of formulas Ia, Ib, Ic, Id, Ie and If, it is stated that the configurations of the UbStitUentS R1A~ R2A~ R3A~ and R4ACH2 in A, the configurations of the æubstituents R1B~ R2B~ R3B~ and R4sCH2 ~ ..
configurations of the substituents Rlc, R2C~ R3C~ and R4CCH2 in C, the configurations of the substituents R1D' R2D' R3D~ and R4DCH2 in D, and the configurations of the substituents R1E' R2~ ~3~, and R4ECH2 in E independently are D-gluco, L-gluco, D-galacto, L-galacto, D-manno, L-manno, D-talo, L-talo, D-allo, L-allo, D-altro, L-altro, D-gulo, L-gulo, D-ido, or L-ido, this is intended to mean that the stereochemical substitution patterns that can be assumed by the various R-groups or R-group-containing groups on the cyclic groups A, B, C, D and E
correspond to the stereochemical patterns formed by the 2-, 3-, and 4-hydroxy groups and the 5-hydroxymethyl group in D-glucose, L-glucose, D-galactose, L-galactose, D-mannose, L-mannose, D-talose, L-talose, D-allose, L-allose, D-altrose, L-alLIvsc, D-gulose, L-gulose, D-idose, or L-idose, respectively.
It will be clear that the ylOUp~ Rl, R2, R3 and CH3 in the group Y are arranged in such a configuration to give a L-galacto-pyranosyl unit and that the group Y therefore is a L-fucose unit or a derivative thereof.
In the compounds of the formula Ia, Ib, Ic, Id, Ie or If, it is preferred that Zl~ Z2~ Z3~ Z4~ Z5~ Z6~ Z7~ Z8~ Zg, Z1O~ Zll~ Z12 Z13, Z14, Z15 and Z16 are 0.

It is also preferred that at the most four, more preferably at the most three, in particular one or two of R1A~ R2A~ R3A~ R4A~
R1B' R2B' R3B~ R4B~ R1C~ R2C, R3C, R4C, R1D~ R2D, R3D, R4D, R1E~
R2Er R3E, or R4E is a group of formula VII.

35 It is also preferred that R1A is a group VII in the ~-configuration.

SUB~ UTE SHEET

W095l00527 1~q961 PCT/SE94/00604 It is also preferred that the configuration f R1A' R2A~ R3A and R4ACH2 in A are D-galacto, A being in the ~-configuration.

Particularly preferred compounds are those wherein R1A is a --5 group VII in the a-configuration and the configuration of R1A' R2A' R3A and R4ACH2 in A are D-galacto, A being in the ~-configuration, especially A is Fuc~1-2Gal~.

It is also preferred that R2~ is Z3, Z5~ Z8~ or Z12~ and the gUration of R1B~ R2B, R3B~ and R4gCH2 in B are D-gluco B
being in the ~-configuration.

It is al~;o preferred that R1B is an acetamido group.

Particularly preferred compounds are those wherein R1A is a group VII in the ~-configuration; the configuration of RlA, R2A ~ ~3A and R4ACH2 in A are D-galacto, A being in the ~-configuration; R2B is Z3, Z5, Z8~ or Z12; and h configuration of R1B~ R2B~ R3B~ and R4BCH2 in B are D-gluco, B
being in the ,B-configuration and R1B is an acetamido group.

Especially interesting are those compounds in which the A-Z3-B
is Fuc~1-2Gal~1-3GlcNAc~ or Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~, those com~oull~s in which A-Z5-B-Z6-C is Fuc~1-2Gal~1-3GlcNAc~1-3Gal~ or Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~, those compounds in which A-Za-B-Zg C-Zlo~D is GalNAc~1-3(Fuc~1-2)Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~ or Fucel-2Ga~l-3(Fuc~l-4)GlcNAc~l-3Gal~l-4Glc~and those cu~ounds in which A-Z12-B-Zl3-c-Zl4-D-zls-E is GalNAc~1-3(Fuc~1-2)Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~l-4Glc~.

It is also preferred that R3B is a group of the formula VII in the ~-configuration.
Particularly preferred compounds are those wherein the Configurations f R1A~ R2A~ R3A~ and R4ACH2 in A and f R1B~ R2B~
R3B~ and R~sBCH2 in B are D-galacto, and the configurations of SU~ UTE SHFET

PCT/SE~/a~O~
W095/00527 2 1 6 4 ~ 6 1.

Rlc~ R2CI R3C, and R4CCH2 in C are D-gluco, A being in the ~-configuration, and B and C being in the ~-configuration, and in which R1B and R3C are groups of the formula VII in the ~-configuration, and in which R1A and R~c are acetamido groups, and R2B iS Z5, Z8 or Z12 ~ and R2C is Z6 ~ Z9 or Z13.

An interesting class of compounds is that in which the carbohydrate moiety contains the structure Y-Zl-A- where Zl is O and the L-fucose unit Y is linked to the 2-position of A.
Examples of interesting basic carbohydrate structures in this class are those having the following formulae where the substituents Rl, R2, R3, R1A~ R2A' R3A' and R4A each indicated as OH, although this is not to be construed as limiting the definitions of the R-substituents in this manner;
rather~ Rl~ R2, R3, R1A~ R2A~ R3A~ and R4A should be considered as being able to assume all the meanings defined above in connection with the formulae Ia, Ib, Ic, Id, Ie and If. Thus, the structure Y-Zl-A- may be Fuc~1-2All~1-Fuc~1-2Alt~1-Fuc~1-2Glc~1-Fuc~1-2Man~l-Fuc~1-2Gul~l-Fuc~1-2Ido~1-Fuc~1-2Gal~1-Fuc~1-2Tal~l-When the groups Rl, R2, R3, R~, RZA' R3A' R4A' R1B' R2B' R3B' R4B~ Rlc~ R2c~ R3c~ R4cr R1D' R2D~ R3D~ R4D~ R1E' R2E~ R3E~ or R4E
in Y, A, B, C, D, and E are not hydroxyl, they may preferably be selected among the following:

H, Cl, F, azido, guanidyl, methyl, ethyl, propyl, vinyl, allyl, prop-l-enyl, ethynyl, prop-2-ynyl, prop-1-ynyl, acetyl, cyclopropyl, cyclopropylmethyl, methoxymethyl, hydroxymethyl, phenyl, oxo, methylene, thiol, amino, methoxy, ethoxy, propoxy, butoxy, hexyloxy, decyloxy, tetradecyloxy, octadecyloxy, vinyloxy, allyloxy, 1-propen-1-yloxy, crotyloxy, SVB~ 111 ~JTE SHE~

O 951~527 1 ~ 1 9 61 ~ PCT/SE94/00604

3-buten-1-yloxy, 2-hexen-1-yloxy, 5-hexen-1-yloxy, S-decen-l-yloxy, 9-decen-1-yl~y, ll-tetradecen-1-yloXy~
oleoyl, ethynyloxy, 2-propyn-1-yloxy, 1-propyn-1-yloxy, methylthio, methylamino, dimethylamino, cyclopropoxy, cyclopropylmethoxy, methoxymethoxy, phenoxy, benzyloxy, 2-furylmethoxy, 2-thienylmethoxy, 2-pyridylmethoxy, trimethylsilyloxy, trimethylsilylethoxy, acetoxy, propionyloxy, butyryloxy, h~xAnoyloxy, decanoyloxy, tetradecanoyloxy, octadecanoyloxy~ acetamido, N-methylacetamido, acetylthio, glycyloxy, or alanyloxy.

Interesting examples of aglycon groups R are the following:
Methyl, ethyl, propyl, isopropyl, butyl, sec.butyl, isobutyl, tert.butyl, pentyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, l-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, heptyl, isoheptyl,

4-methylhexyl, 3-methylhexyl, 2-methylhexyl, 1-methylhexyl, 3-ethylpentyl, 2-ethylpentyl, l-ethylpentyl, 1-propylbutyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, tetracosyl, cyclG~ro~yl, cyclopropylethyl, cyclobutyl, cyclobutylmethyl, cyclopentylmethyl, cyclopentylprop-3-yl, cyclohexyl, cyclohexylmethyl, cyclohexylprop-3-yl, cycloheptyl, phenyl, 4-nitrophenyl, benzyl, 4-phenylprop-1-yl, 3-hexylthio-2-(hexylthio)methylprop-1-yl, 3-hexylsulfonyl-2-(hexylsulfonyl)methylprop-1-yl, 3-decylthio-2-(decylthio)methylprop-1-yl, 3-decylsulfonyl-2-(decylsulfonyl)methylprop-1-yl, 8-amino-3l6-dioxaoct-1-yl, 1,3-dihydroxyprop-2-yl, 1,3-diaminoprop-2-yl, 3-hydroxy-2-(hydroxymethyl)prop-1-yl, 2-phenylthioethyl or trimethylsilylethyl.

In a group R comprising a matrix MA, the linkage between the matrix MA and the remainder of R may typically be through any of the spacers well known in the field of protein conjugates, cf. for example J.H. Pazur, Adv. Carbohydr. Chem. Biochem., Vol 39, (1980), 40S-447; Y.C. Lee & R.T. Lee, "Glycoconjugates~, Vol. 4 Part B, 57-83, Ed. Horowitz, Academic Press, N.Y.
(1982); and G. Magnusson, FEMS Symposium, 215-228 (1986). In SUB~ 11- ~JTE SHEEt' W095/00527 21 ~ ~ 9 G 1 --7 6-- PCT/SE94/006~

the present context, the term "Spacer" is intended to mean a molecule moiety which links the active substance to a carrier.
A spacer molecule is designed to have two different ~unctionalities, each reacting specifically with another functionality, a linear moiety being placed between these two functionalities. By li~ki~g the active substance to a carrier v~a a Spacer, one makes the active substance more accessible, e.g. to H. pylori adhesins or colonization factor antigens.

The Spacer can be defined as (W)v-S'-P', wherein S' is an Cl_ alkyl~ an C2-24 alkenyl, an Cl_24alkylaryl, an arylCl_24alkyl an arylC1_24alkylaryl, an Cl_24alkylarylcl-24alkyl group which groups may be interrupted by carbonyl, thiocarbonyl, oxycarbonyl, carbonyloxy, carbonylamino, aminocarbonyl, aza, oxa or ~hia groups; an aryl group, an aryloxy, an C1_24alkoxy, a polyethyleneglycol group, a steroid group, a sphingoid group;
all groups may be substituted with carboxyl, Cl_4alkylcarbonyl, amide, hydroxy, alkoxy, aryloxy, phenoxy;

P' is NH--C(S), NH--C(O), C(O), NH, C(S), C(O)O, (O)CO, SO, SO2, SO3, SO4, PO3, PO4;
W iS N~-C(S), NR-C(O), C(O), C(S), C(O)O, (O)CO, SO, S02, SO3, SO4, PO2, PO3 ~ PO4 ' with the proviso that when Zl~ Z2 ~ Z4 ~ Z7 ~ Zll or Z16 are CH2 then W cannot be P02, with the proviso that when Zl~ Z2~ Z4~ Z7~ Zll or Z16 are O or S
then W cannot be ~O)CO, SO4 or PO4, and with the proviso that when Zl, Z2~ Z4~ Z7~ Z11 or Z16 are NH then W cannot be NH-C(S), . NH-C(O) ~ (O)CO, SO4, PO4; and v is an integer O or 1.
The atom of the sugar moiety which linkages to the spacer is selected among from the following: -O-, -S-, -NH-, -CH2-preferably -O-.

In the compounds of the formulas Ia, Ib, Ic, Id, Ie and If, the various groups R carrying the matrix MA may themselves comprise the spacer and the linkage. Specific and typical examples of linkages are those formed through amino group- or keto group-SUB~ JTE SHEEI-W095/00~7 21 6 ~ 9 ~1 PCT/SE94100604 containing matrices.: Such linkages between the spacer and the matrix may have the following general structures:

-NH-C(S)N~- or -C(O) -NH-, or -NH-C(OJ--` 5 wherein ~he atoms marked bold and italic orginate from the given matrix.

The number of structures of the formulas Ia, Ib, Ic, Id, Ie or If on each matrix unit may be mono- or multivalent and may vary between ~ to lO,OOO, depending on the nature of the matrix.

Below follow a non-limiting list of examples of spacers suitable between Q and the remainder of R

spacer 1 ~~~~~

spacer 2 ~~ ~~ O~'~

OH CH~

spacer 3 ; ~H~\ ~ Cl 1~1 ~ Nl;

spacer 4 ~ spacer 5 ~ ~ ~

In the li~t of spacers given above and below, the atoms marked in bold italics originate from the matrix in question.
The vertical wavy lines on the left and right ends in the spacer above signify that there are bonds at the ends.
As examples of compounds of the general formulas Ia, Ib, Ic, Id, Ie or If comprising matrix moieties, the following may be mentioned-SUB:~ 111 UTE SHEET

WO 95/00527 216 4 9 6 I PCT/SE94/00604~

~0~ --~NHCH~-BSA
' m' O

--O ~ ~ NH C-polyacrylam~d~

. ..~.

-o \ ~NH C-poJ~c~JJm;d~

\~o~ <~ OH
H O ~ cHzNHc~ ~cr~l~mld~
~.~ OH

H ~
- O~ - OH
HO~ ~-- --C~NH-N~-~S~
H OH

-\~o~ f' I ~ ~Jl( ~ GSA

SUE3~ JTE SHEET

WO 95/00527 21 6 ~ PCT/SEg4/00604 ~0 (-CH2 )p CNH-8SA

~_ O (-CH2-)p ~--C--NH-HSA

.0 0 \ ~_O (-CH2-)p--~--C--PAA

~~ \ o_ ~_8 ~o~ ~_C_NH-HSA

--N _ 8~o --N - C - PAA

~ ~0~

~ ~C~
O

SUB~ 111 ~JTE SHEET

wogs/~7 PCT/SE94/00604 2`1G`~9 6~ -20-When ~ is used in the examples above, this has the meaning of mono-, di-, tri- or oligosaccharide as specified in the text, and m' is an integer 0-5 and p is an integer 0-13.
When the matrix above is exemplified by BSA, HSA and polyacrylamide(PAA) this can be any other protein or peptide or other matrix specified in the text.

Specific examples of interesting compounds of the formula Ia, Ib, Ic, Id, Ie or If are the following:.
Fuc~1-2Gal~l-O-propyl Fuc~1-2Gal~1-O-iso~r~pyl Fuc~1-2Gal~l-O-butyl Fuc1-2Gal~l-O-tert-butyl Fuc~1-2Gal~l-O-hexyl Fuc~l-2Gal~l-O-octyl Fuc~1-2Gal~l-O-decyl Fuc~1-2Gal~l-O-tetradecyl Fuc~1-2Gal~l-O-octadecyl Fuc~l-2Gal~1-O-(C6bissulfide) Fuc~1-2Gal~l-o-(C1Obissulfide) Fuc~1-2Gal~l-O-(C6bissulfone) Fuc~1-2Gal~l-O-(ClObissulfone) Fuc~1-2Gal~l-O-(8-amino-3,6-dioxaoct-1-yl) Fuc~1-2Gal~1-3GlcNAc~l-O-propyl Fuc~1-2Gal~1-3GlcNAc~l-o-isopropyl Fuc~1-2Gal~1-3GlcNAc~l-O-butyl Fuc~1-2Gal~1-3GlcNAc~l-O-tert.butyl Fuc~1-2Gal~1-3GlcNAc~l-O-hexyl Fuc~1-2Gal~1-3GlcNAc~1-O-octyl Fuc~1-2Gal~1-3GlcNAc~l-O-decyl 35 Fuc~1-2Gal~1-3GlcNAc~l-O-tetradecyl Fuc~1-2Gal~1-3GlcNAc~1-O-octadecyl Fuc~1-2Gal~1-3GlcNAc~-1-O-(C6bissulfide) SlJ~ .ITE SHEET

wos5l0os27 Q PCT/SE94/00604 _2~ 6~`~Gl Fuc~1-2Gal~1-3GlcNAc~-1-O-(C6bissulfone) Fuc~1-2.al~1-3GlcNAc~-1-0-(8-amino-3,6-dioxaoct-1-yl) Fuc~1-2Gal~1-3Glc~l-O-propyl Fuc~1-2Gal~1-3Glc~1-O-isopropyl Fuc~1-2Gal~1-3Glc~1-O-butyl Fuc~1-2Gal~1-3Glc~1-O-tert.butyl Fuc~1-2Gal~1-3Glc~1-O-hexyl Fuc~1-2Gal~1-3Glc~l-O-octyl Fuc~1-2Gal~1-3Glc~1-O-tetradecyl Fuc~1-2Gal~1-3Glc~1-O-octadecyl Fuc~l-2Gal~l-3Glc~1-O-(C6bissulfide) Fuc~1-2Gal~1-3Glc~1-O-(C6bissulfone) Fuc~1-2C~al~1-3Glc~1-O-(8-amino-3,6-dioxaoctyl) wherein C6bissulfide - 3-hexylthio-2-(hexylthio)methylprop-1-yl-C~Obissulfide ~ 3-decylthio-2-(decylthio)methylprop-1-yl-C6bissulfone ~ 3-hexylsulfonyl-2-(hexylsulfonyl)methylprop --1--yl--ClObissulfone ~ 3-decylthio-2-(decylthio)methylprop-1-yl-. 25 Further interesting compounds are:

Fuc~1-2Gal~1-O-Me Fuc~1-3Glc~1-O-Me Fuc~1-3GlcNAc~1-O-Me Fuc~1-3GlcNAc~l-Spacer l-BSA
Fuc~1-3GlcNAc~l-O tetradecyl Fuc~1-4GlcNAc~l-O-Me Fuc~1-4GlcNAc~1-Spacer 2-polyacrylamide Fuc~1-4GlcNAc~1-O-tetradecyl Fuc~1-4Gal~l-o-Me Fuc1-6Gal~l-O-Me Fuc~1-6Gal~1-Spacer 2-polyacrylamide Fuc~1-2Gal~l-Spacer 2-polyacrylamide SUB~ ~ JTE SHEET

wo g5,00527 2 1 ~ 4 ~ 6 ~ -22- PCT/SEg4/00604 Fuc~1-2Gal~l-Spacer-1-BSA
Fuc1-2Gal~l-Spacer 1-HSA
Fuc~1-2Gal~1-Spacer 4-BSA
Fuc~1-2Gal~1-Spacer 4-HSA
Fuc~1-2Gal~l-spacer s-polyacrylamide .
Fuc~1-2Gal~1-0-tetradecyl Fuc~1-2Gal~1-3GlcNAc~1-Spacer 5-polyacrylamide Fuc~l-2Gal~l-3Glc~Ac~l-spacer 4-BSA
Fuc~1-2Gal~1-3GlcNAc~l-Spacer 4-HSA
Fuc~l-2Gal~l-3GlcNAc~l-spacer 2-polyacrylamide Fuc~l-2Gal~l-3GlcNAc~l-spacer 1-HSA
Fuc~l-2Gal~l-3GlcNAc~l-spacer 1-~SA
Fuc~l-2Gal~l-3GlcNAc~l-o-tetrade Fuc~1-2Gal~1-3Glc~1-Spacer 1-HSA
Fuc~1-2Gal~1-3Glc~1-Spacer 1-BSA
Fuc~l-2Gal~l-3Glc~l-spacer 4-HSA
Fuc~1-2Gal~1-3Glc~1-spacer 4-BSA
Fuc~l-2Gal~l-3Glc~l-spacer 2-polyacrylamide Fuc~l-2Gal~l-3Glc~l-spacer s-polyacrylamide Fuc~1-2Gal~1-3(Fuc~1-4)Glc~1-Spacer 1-HSA
Fuc~1-2Gal~1-3(Fuc~1-4)Glc~1-Spacer 1-BSA
Fuc~1-2Gal~1-3(Fuc~1-4)Glc~1-Spacer 4-HSA
Fuc~1-2Gal~1-3(Fucal-4)Glc~l-Spacer 4-BSA
Fuc~1-2Gal~1-3(Fuc~1-4)Glc~1-Spacer 2-polyacrylamide Fuc~1-2Gal~1-3(Fuc~1-4)Glc~l-Spacer 5-polyacrylamide Fucel-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-Spacer 3-BSA
Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-Spacer 2-polyacrylamide Fuc~1-2Gal~1-3(Fuc~1-4)GlcN~c~1-3Gal~1-Spacer 5-polyacrylamide Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-0-tetradecyl Fuc~1-2Gal~1-4Glc~1-Spacer l-BSA
Fuc~1-2Gal~1-4Glc~1-Spacer 2-polyacrylamide Fuc~1-2Gal~1-4Glc~l-0-tetradecyl Gal~1-4(Fuc~1-3)GlcNAc~1-Spacer 1-BSA
Gal~1-4(Fuc~1-3)GlcNAc~1-Spacer 2-polyacrylamide Gal~1-4(Fuc~1-3)GlcNAc~1-O-tetradecyl Fuc~1-2Gal~1-3GlcNAc~1-3Gal~1-Spacer 3-BSA
Fuc~1-2Gal~1-3GlcNAc~1-3Gal~1-Spacer 3-HSA
Fuc~1-2Gal~1-3GlcNAc~1-3Gal~1-Spacer 5-polyacrylamide SUB~ I ~ ~ LITE SHEET

Wog5/00~7 -23~ PCT/SE94/00604 ~Uc~l-2Gal~l-3GlcNAc~l-3Gal~l-spacer l-HSA
Fuc~1-2~al~1-3GlcNAc~1-3Gal~l-Spacer 5-BSA
Fuc~1-2Gal~1-3GlcNAc~1-3Gal~l-Spacer 4-HSA
Fuc~1-2Gal~1-3GlcNAc~1-3Gal~1-Spacer 4-BSA
Fucal-2~al~1-3GlcNAc~1-3Gal~l-Spacer 2-polyacrylamide Fuc~1-2Gal~1-3GlcNAc~1-3Gal~l-o-tetradecyl GalNAc~1-3~Fuc~l-2)3Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~l-Spacer-GalNAc~1-3(Fucal-2)3Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~l-Spacer-2-polyacrylamide GalNAcal-3tFuc~1-2)3Gal~l-3(Fucc1-4)GlcNAc~1-3Gal~1-0-tetra-decyl Fucal-2~al~1-4(Fuc~1-3jGlc~l-Spacer l-BSA
Fucal-2Gal~1-4(Fuc~1-3)Glc~l-Spacer 2-polyacrylamide 15. Fucal-2Gal~1-4(Fuc~1-3)Glc~l-0-tetradeCyl Fucal-2(3-o-methyl)Gal~1-0-tetradecyl ~UC~1-2(3-O-methyl)Gal~l-Spacer 1-BSA
Fucal-2(3-o-methyl)Gal~1-spacer 2-polyacrylamide Fucal-2(3-o-allyl)Gal~1-spacer 1-BSA
Fucal-2(3-o-allyl)Gal~l-spacer 2-polyacrylamide Fucal-2(3-O-allyl)Gal~1-0-tetradecyl Fucal-2(3-o-propyl)Gal~l-Spacer l-HSA
Fucal-2(3-O-propyl)Gal~l-Spacer l-BSA
Fuc~l-2(3-o-propyl)Gal~l-spacer 2-polyacrylamide ~ucal-2(3-O-propyl)Gal~l-Spacer 4-HSA
Fucal-2(3-o-propyl)Gal~l-Spacer 4-BSA
Fucal-2t3-O-propyl)Gal~l-Spacer 5-polyacrylamide Fucal-2(3-O-butyl)Gal~l-Spacer 1-BSA
Fucal-2(3-o-butyl)Gal~l-Spacer 2-polyacrylamide Fucal-2(3-O-butyl)Gal~l-0-tetradecyl Fucal-2(3-o-methyl)Gal~1-3GlcNAc~l-Spacer l-BSA
Fuc1-2(3-o-methyl)Gal~1-3GlcNAc~l-Spacer 2-polyacrylamide . Fucal-2(3-0-methyl)Gal~1-3GlcNAc~l-0-tetradecyl Fucal-2(3-o-allyl)Gal~1-3GlcNAc~l-Spacer l-BSA
Fuc~1-2(3-O-allyl)Gal~1-3GlcNAc~l-Spacer 2-polyacrylamide Fucal-2(3-o-allyl)Gal~1-3GlcNAc~l-0-tetradecyl Fucal-2(3-o-propyl)Gal~1-3GlcNac~l-Spacer l-HSA
Fucal-2t3-o-propyl)Gal~l-3GlcNac~l-Spacer l-BSA

SUB~ JTE SHEEr WO 95/~527 216 ~ 9 6 I PCT/SE94100604 Fucal-2(3-O-propyl~Gal~1-3GlcNac~1-Spacer 2-polyacrylamide Fuc~1-2(3-O-propyl)Gal~1-3GlcNac~1-Spacer 4-HSA
Fuc~1-2(3-O-propyl)Gal~1-3GlcNac~1-SpaCer 4-BSA
Fucal-2(3-o-propyl)Gal~1-3GlcNac~1-Spacer 5-polyacrylamide Fucal-2(3-O-butyl)Gal~1-3GlcNAc~1-Spacer 1-BSA ..
Fucal-2(3-o-butyl)Gal~l-3GlcNAc~l-spacer 2-polyacrylamide Fucal-2(3-O-butyl)Gal~1-3GlcNAc~l-O-tetradecyl Fucal-2(3-O-propyl)Gal~1-3(Fucal-4)GlcNac~l-Spacer l-HSA
Fucal-2(3-o-propyl)Gal~1-3(Fucal-4)GlcNac~l-SpaCer l-BSA
Fucal-2(3-O-propyl)Gal~1-3(Fuc~1-4)GlcNac~l-Spacer 2-polyacrylamide Fucal-2(3-o-propyl)Gal~1-3(Fucal-4)GlcNac~l-Spacer 4-HSA
Fucal-2(3-o-propyl)Gal~1-3(Fuc~1-4)GlcNac~l-Spacer 4-BSA
Fucal-2(3-o-propyl)Gal~1-3(Fucal-4)GlcNac~1-Spacer 5-polyacrylamide Fucal-2(3-O-methyl)Gal~1-4GlcNAc~l-Spacer 1-BSA
Fucal-2(3-O-methyl)Gal~1-4GlcNAc~1-Spacer 2-polyacrylamide Fuc1-2(3-O-methyl)Gal~1-4GlcNAc~l-O-tetradecyl Fucal-2(3-O-allyl)Gal~1-4GlcNAc~1-Spacer 1-BSA
Fucal-2(3-O-allyl)Gal~1-4GlcNAc~1-Spacer 2-polyacrylamide Fucal-2(3-O-allyl)Gal~1-4GlcNAc~1-O-tetradecyl Fuc~1-2(3-O-butyl)Gal~1-4GlcNAc~1-Spacer l-BSA
Fucal-2(3-O-butyl)Gal~1-4GlcNAc~l-Spacer 2-polyacrylamide Fucal-2(3-O-butyl)Gal~1-4GlcNAc~l-O-tetradecyl Fucal-2(3-O-methyl)Gal~1-3Glc~1-Spacer 1-BSA
Fucal-2(3-o-methyl)Gal~1-3Glc~l-Spacer 2-polyacrylamide Fucal-2(3-O-methyl)Gal~1-3Glc~1-O-tetradecyl Fucal-2(3-O-allyl)Gal~1-3Glc~1-Spacer 1-BSA
Fucal-2(3-O-allyl)Gal~1-3Glc~l-Spacer 2-polyacrylamide Fucal-2(3-O-allyl)Gal~1-3Glc~l-O-tetradecyl Fuc~1-2(3-O-butyl)Gal~1-3Glc~l-Spacer l-BSA
Fucal-2(3-O-butyl)Gal~1-3Glc~l-Spacer 2-polyacrylamide Fucal-2(3-O-butyl)Gal~1-3Glc~l-O-tetradecyl Fucal-2Gal~1-4GlcNAc~1-Spacer 1-BSA
Fuc~1-2Gal~1-4GlcNAc~l-spacer 2-polyacrylamide Fuc~1-2Gal~1-4GlcNAc~l-O-tetradecyl Gal~1-3(Fucal-2)Gal~l-Spacer 1-BSA
Galal-3(Fucal-2)Gal~l-Spacer 2-polyacrylamide SUB~ JTE SHEEr wo 95/00s27 ~?1 6~9 S PCT/SEg4/00604 Gal~1-3tFuc~1-2)Gal~1-0-tetradecyl GalNAc~l-3(Fuc~l-2)Gal~l-4Glc~l-spacer 1-BSA
GalNAcel-3(Fuc~l-2)Gal~l-4Glc~l-spacer 2-polyacrylamide GalNAcal-3(Fuc~1-2)Gal~1-4Glc~1-0-tetradecyl Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-4Glc~1-Spacer 1-BSA
Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-4Glc~l-Spacer 2-polyacrylamide Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-4Glc~1-0-tetradecyl Gal~1-3(Fuc~1-4)GlcNAc~l-Spacer l-BSA
Gal~1-3(Fuc~1-4)GlcNAc~l-Spacer 2-polyacrylamide Gal~1-3(Fuc~1-4)GlcNAc~1-0-tetradecyl In the present application, such as the list above, specific compounds or parts of compounds may be named or represented in a condensed form corresponding to the recommendations concerning nomenclature of glycoproteins, glycopeptides, and peptidoglycans made by the Joint Commission on Biochemical Nomenclature under the International Union of Pure and Applied Chemistry and the International Union of Biochemistry (cf. Pure & Applied Chem., Vol. 60, No. 9, pp 1389-1394, 1988).
In another aspect, the invention concerns a pharmaceutical composition comprising a compound of the formula Ia, Ib, Ic, Id, Ie or If as defined above or a mixture thereof in combination with at least one anti-ulcer medicament, or with at least one antibacterially active compound, or mixtures thereof, as well as a pharmaceutically acceptable carrier.

The term "anti-ulcer medicament" is intended to denote any substance or composition which is able to reduce or participate in reducing gastrointestinal ulcerations, in particular ulcerations in the stomach or duodenum. Pharmaceutical compositions according to the invention containing such substances or compositions have the potential advantage of being able to provide a dual effect by on the one hand reducing the ulceration and on the other hand simultaneously lowering the degree of infection in the stomach by H. pylori by preventing or inhibiting the adhesion of the bacterium onto the gastric or duodenal mucosa, thereby further promoting the SUB~ 111 ~JTE SHEET~

.

Wogsl~7 2 1 ~ ~ 9 6 I PCT/SE94/00604 healing of an ulcer; Suitable types of anti-ulcer medicaments are gastric secretion inhibiting compounds (primarily acid secretion inhibiting compounds) and antacids.

In a preferred aspect of the use according to the invention, the pharmaceutical composition prepared is adapted to be administered in combination with a preparation for s~ Ard therapy of gastritis or ulcus, such as preparations containing anti-ulcer or anti-gastritis medicaments, e.g. selected among gastric secretion inhibiting compounds such as omeprazole, - cimetidine, ranitidine, lansoprazole, pantoprazole, sucralfate, famotidine, or nizatidine, or antacids such as magnesium hydroxide, aluminium hydroxide, calcium carbonate, sodium carbonate, sodium hydrogen carbonate, simethicone or aluminium magnesium hydroxide or a hydrate thereof (such as the monohydrate known as magaldrate).

In another preferred aspect of the use according to the invention, the pharmaceutical composition prepared is adapted to be administered in combination with a preparation for a course of therapy with an antibacterial agent, such as an antibacterial agent selected from those listed above, in particular preparations containing ~-lactam antibiotics such as amoxicillin, ampicillin, cephalothin, cefaclor or cefixime; or macrolides such as erythromycin, or clarithromycin; or tetracyclines such as tetracycline or doxycycline; or aminoglycosides such as gentamycin, k~A~ycin or amikacin; or quinolones such as norfloxacin, ciprofloxacin or enoxacin; or - others such as metronidazole, nitrofurantoin or chloramphenicol; or preparations containing bismuth salts such as bismuth subcitrate, bismuth subsalicylate, bismuth subcarbonate, bismuth subnitrate or bismuth subgallate.

In a further aspect, the invention concerns all novel compounds 2mong those having the formula Ia, Ib, Ic, Id, Ie or If defined above.

SU~ .JTE SHEET

WOs5/00~7 -27- 2 1 ~ ~ 9 6 1 PCT/SE94100604 The compounds of formula Ia, Ib, Ic, Id, Ie or If can be prepare~ according to several general methods using monosaccharides or oligosaccharides as starting materials.
Functional group transformations can be performed before or S after the formation of glycoside bonds. To ensure transformations of the functional group in a certain position, the use of reactions which are regiospecific or the protection with protective yLoup~ may optionally be neceRs~ry. The protective y~OU~S can be removed or can form part of the compoun~ in question.

The compounds of the invention can e.g. be prepared as shown in the scheme below. In the scheme, al~ho~gh specific substit~ents or configuration may be shown, it is to be lS understood that to the extent that it is appropriate, the various groups shown may assume the full varia~ility range as defined for the general formulae Ia, Ib, Ic, Id, Ie, and If.

S~IB~ ITE SHEET

L96~ ~

MONOSACCHARIDES

Step 1 H~\~ Ra ~\ ~Ra ~ c OH OH

Step 2 H~\~ Ra ~ ~ Rs Rb Rb Step 3 CH3 ~o ~< ~~ ~~
HO \ ~~ \ ~z Ra Rb /n \ Rb / m Step 4 CH3 ~ o ~< ~~
HO \ ~~ \ ~z Rc Rb /n \ /m SUB~ I 11 ~JTE SHEET

2ls~96~
wos~loQ~27 PCT/SE94/00604 In the first step (step 1) a monosaccharide, e.g. L-fucose, D-galactose, D-glucose, 2-deoxy-2-phthalimido-D-glucose, 2-deoxy-2-phthalimido-D-galactose~ D-mannose, is converted to a glycoside, with aglycons (Ra), e.g. SEt, SPh, OTMSEt, O-allyl or OBn (~nown aglycons in the art), to form the Ra-glycoside derivati~e in such a way that the Ra-glycoside is possible to ~ transfor~ to a glycosyl donator by activation of the anomeric centre. 'rhe Ra-glycosides can be prepared as follows: A
monosacc~aride as above is per-O-acylated with acetic anhydride in pyridine or with acetic anhydride-sodium acetate or with benzoyl chloride in pyridine. The monosaccharide per-O-acylate is reacted with, e.g. hydrogen bromide or hydrogen chloride in a suitable solvent such as, e.g. acetic acid or dichloro~ethane~ to form per-O-acylated glycosyl bromide or chloride (e.g. on O-acylation and glycosyl halide synthesis, see M. L. Wolfrom and A. Thompson, Methods in Carbohydrate Chemistr~, Vol. 2, 211-215, edited by R. L. Whistler and M. L.
Wolfrom, Academic Press, New York, 1963, G. Hewit and G.
Fletcher Jr., ibid, 226-228, and R. U. Lemieux, ibid, 223-224).
The aglycon (Ra) is trans~erred to the monosaccharide by reacting a suitable thio~ or alcohol, e.g. HSEt, HSPh, HOTMSEt, HO-allyl, or HOBn with the monosaccharide per-O-acylate using a Lewis acid such as boron trifluoride etherate (see e.g. R. J.
Ferrier and R. H. Furneaux, Car~ohydr. Res. 52 (1976), 63-68, J. Dahmén, T. Frejd, G. Gronberg, T. Lave, G. Magnusson, and G.
Noori, Carbohydr. Res. 116 (1983), 303-307), or trimethylsilyl trifluoromethanesulfonate (see T. Ogawa, K. Beppu, S.
N~k~h~yashi, Carbohydr. Res. 93 (1981), C6-C9) as promoters.
The reac~ion is carried out in a suitable solvent such as chloroform, dichloromethane and/or toluene. When the monosaccl~aride derivative in question is a per-O-acylated glycosyl bromide or chloride, promoters such as silver trifluoromethanesulfonate or mercury(II) salts (see e.g. H.
Paulsen, Angew. Chem. Int. Ed. Engl. 21 (1982), 155-173) can be . 35 used, and the reactions are carried out in suitable solvents such as dichloromethane and/or toluene. The monosaccharide Ra-glycosides is obtained after de-O-acylation using sodium methoxide (see e.g. A. Thompson, M. L. Wolfrom, and E. Pascu, SUB~ JTE SHEET

W095l00527 ~16 ~ 9 ~1 pcTlæs4loo6o4~

Methods in Carbohydrate Chemistry, Vol. 2, 215-220, edited by R. L. Whistler and M. L. Wolfrom, Academic Press, New York, 1963) in methanol or in methanol containing a co-solvent such as dichloromethane or tetrahydrofuran.
In the second step (step 2) the monosaccharide Ra-glycoside is ~urther derivatized. New functional groups (Rb) which will form part of the ~inal product or act as protective groups during the subsequent glycosylation steps are introduced. Examples of functional group transformations are: OH-groups to ethers or esters (see e.g. Protective Groups in Organic Synthesis edited by T. W. Greene and P. G. M. Wuts, John Wiley & Sons, Inc., New York, 1991), OH-groups to carbonates (see e.g. J. March, Advanced Organic Chemistry - Reaction MechAni-cms~ and Structure, 347, 3rd Ed., John Wiley & Sons, New York, 1985, and references cited herein), reductive removal or OH-groups via halides, sulfonates or other routes (see e.g. J. March, Advanced Organic Chemistry - Reaction ~eC~A~ i -sms, and Structure, 389-392, 394, 3rd Ed., John Wiley & Sons, New York, 1985, and references cited herein, and H. H. Baer, Pure Appl.
Chem. 61~7) (1989), 1217-1234, and references cited herein), OH-groups to halogen (see e.g. J. March, Advanced Organic Ch~ çtry - Reaction NechAnicms~ and Structure, 381-286, 3rd Ed., John Wiley & Sons, New York, 1985, and references cited herein), OH-groups to azido groups (see e.g. J. March, Advanced organic Chemistry - ~eaction Mech~ni~cms~ and Structure, 380, 3rd Ed., John Wiley & Sons, New York, 1985, and references cited herein, and H. H. Baer, Pure Appl. Chem. 61~7) (1989), 1217-1234, and references cited herein), OH-groups to amino groups via azides or other routes (see e.g. J. March, Advanced Organic ~ri-ctry - Reaction MechAnicms~ and Structure, 798-800. 1106, 3rd Ed., John Wiley & Sons, New York, 1985, and references cited herein, and H. H. Baer, Pure Appl . Chem . 61 ( 7 ) (1989), 1217-1234, and references cited herein), OH groups to keto groups (oxo) (see e.g. J. March, Advanced Organic Chemistry - ~eaction Mec~An;~ms, and structure, 1048-1120, 3rd Ed., John W~ley & Sons, New York, 1985, and references cited herein). OH groups to exomethylene derivatives via keto groups SU~ JTE SHEEr W095/~52Y ~9 6 ~31- PCT/SE94/00604 or other routes (see e.g. J. March, Advanced Organic Chemistry - ~eact~on ~e~hAn;sms, and Structure, 400-404, 407, 845-854, 3rd Ed. r John Wiley & Sons, New York, 1985, and references cited herein), OH groups to alkyl groups via exomethylene derivatives and subsequent hydrogenation or via other routes (æee e.g. H. O. H. House, Modern synthetic Reactions, 1-130, 2nd Ed.~ W. A. Benjamin, Inc., Menlo Park, C.A., 1972, and references cites herein, or J. Yoshimura, Adv. Car~ohydr. Chem.
BiochemO ~2 il984), 69-134), and PYchA~ge of OH groups for heterocyclie ~GU~D via different routes (see e.g. A. R.
Xatrizky, Handbook of Heterocyclic Chemistry, Pergamon Press, Oxford, 1985).
.
In the third step (step 3), condensation of the Ra-glycosides substituted with functional ~L OU~ (Rb) (protective groups known inn the ar~) from a~ove are performed. For O-glycosidic linkages: One Ra-glycoside derivative is transformed to a glycosyl donor by activation at the anomeric centre, and reacted with another Ra-glycoside which has been transformed to a glycosyl acceptor by removing one or several protective ~L OU~ ( see e.g. H. Paulsen, Angew. Chem. Int. Ed. Engl. 21 (1982), 155-173, R. R. Schmidt, Angew. Chem. Int. Ed. Engl. 25 (1986), 212-235, P. F~gedi, P. J. Garegg, H. Lonn, and T.
Norberg, Glycoconj. J. ~ (1987), 97-108, Protective Groups in Organic Synthesis edited by T. W. Greene and P G. M. Wuts, John Wiley & Sons, Inc., New York, 1991). For C-glycosidic linkages see e.g. R. R. Schmidt, and G. Effenberger, Liebigs Ann. Chem.
(1987), 82S-831, S. Czernecki, and G. Ville, J. Org. Chem. 5 (1989), 610-612, R. Preuss, and R. R. Schmidt, J. Carbohydr.
Chem. 10l5) (1992), 887-900, o. Martin, and W. Lai, J. Org.
Chem. 5~ (1993), 176-185, or C. R. Bertozzi, P. D. Hoeprich, Jr., and M. D. Bednarski, J. Org. Chem. 57 (1992), 6092-6094.
For S-glycosidic linkages see e.g. L-X Wang, N. Sakairi, and H.
Kuzuhars, J. Chem. Soc. Perkin ~rans. 1 (1990), 1677-1982, or M. Blanc-Meusser, L. Vigne, H. Driguez, J. Lehman, J. Streck, and K. Urbahns, Carbohydr. Res. 22~ (1982), 59-71.

SUB~ JTE SHEEi-W095/~27 2 ~ 6 ~ 32- PCT/SE94/00604 Further glycosidic linkages may be introduced by repeating the third step.

In the fourth step (step 4) the substituent (Rc) at the reducing end is introduced. Rc is defined as (Zl-z16)-~ ~
wherein R and Zl-z16 have the definition given for compounds Ia, Ib, Ic, Id, Ie and If. The term "(Zl-Z16)-R" shall be read as Zl-R, 22-~, Z3-~...... Z16-~- Activation of an oligosaccharide Ra-glycoside derivative from step 3 at the anomeric centre of the reducing end and reaction with a suitable nucleophile leads to O-, C-, S-, or N-glycosidic derivatives, respectively. A final product i5 obtained after removal of protective groups, if necessary. When the compound o~ the invention is in the form of a conjugate with a particular matrix, the Rc-glycoside derivative is further transformed via different routes to the final product (see e.g.
Y. G. Lee, and R. T. Lee, Glycoconjugates, 121-164, edited by H. J. Allen, and E. C. Kisailus, Dekker, New York, 1992, R.
Roy, F. D. Tropper, and A. Romanowska, J. Soc., Chem. Commun.
(1992), 1611-1613, or C. P. Sotwell and Y. C. Lee, Adv.
Carbohydr. Chem. Biochem. , Vol. 37 tl980), 225-281).

Copolymerisation reactions for preparation o~ copolymers of acrylamide and the mono-, di-, tri- or oligosaccharide glycosides with or without a spacer are performed by known methods, for example as described in E. Xallin, H. Lonn, T.
Norberg and M. Elofsson, J. Carbohydr. Chemistry 8(4), 597-611 (1989) or ~. Andersson and S. Oscarsson, Bioconjugate Chemistry, vol. 4(3), 246-247 (1g93). The general strategy for preparation of these conjugates has been to attach an olefinic yL~ to a carbohydrate, and then copolymerize this derivative with acrylamide. The olefinic group has been introduced into the car~ohydrate molecule either as an allyl glycoside at an early stage by acryloylation of an amino function of a mono-, di-, tri- or oligosaccharide derivative or by other known methods.

SIJB~ JTE SHEET

W095/~52~ 21 6 ~ g 6 1 PCT/SE94/00604 As indicated above, pharmaceutical preparations containing the compou~ds of the general formula Ia, Ib, Ic, Id, Ie or If constitute a further aspect of the invention.

The compounds of the invention can be administered systemically or locally and are preferably administered orally or by injection, by the rectal route, by the transdermal route, by infusion or by inhalation in the form of a pharmaceutical preparation comprising the active ingredient in the form of the original compound or in the form of a pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable carrier which may be a solid, semi-solid or liquid diluent or an ingestible capsule, and such preparations compri e a further aspect of the invention. Pharmaceutically acceptable carriers must, of course, be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to human or mammals being treated. The compounds may also be used without carrier material. As examples of pharmaceutical preparations may be mentioned tablets, capsules, dragees, solutions, drops, such as nasal ~s, aerosols for inhalation, nasal spray, liposomes, etc.
Usually the active substance will comprise between O.Ol and 99 % by weight of the preparation, e.g. between 0.5 and 20% by weight for preparations intended for injection and between O.l and So~ by weight for preparations intended for oral administration.

~he preparations are preferably in unit dosage form, whether as single dosage units or as multiple dosage units.
To produce pharmaceutical preparations in the form of dosage units for oral application containing a compound of the invention, the active ingredient may be mixed with conventionally used solids, pulverulent carriers, e.g. lactose, 35 saccharose, sorbitol, mannitol, a starch such as potato starch, corn starch, amylopectin, laminaria powder or citrus pulp powder, a cellulose derivative or gelatine and also may include lubricants such as magnesium or calcium stearate or a Carbowax~

SUB~ ITE SHEFJ--WO95/~527 216 4 9 61 pcTJsæ94loo6o4 or other polyethylene glycol waxes and compressed to form tablets or cores for drag~es. If drag~es are required, the cores may be coated with e.g. concentrated sugar solutions which may contain gum arabic, talc and/or titanium dioxide, or , alternatively~ with a film forming agent dissolved in easily volatile organic solvents or mixtures of organic solvents.
Dyestuffs can be added to these coatings, e.g. to distinguish between different contents of active substance. For the preparation of soft gelatine capsules consisting of gelatine and, e.g. glycerol and a plasticizer, or similar closed capsules, the active substance may be admixed with a Carbowax~
or a suitable oil such as e.g. sesame oil, olive oil, or arachis oil. Hard gelatine capsules may contain granulates of the active substance with solid, pulverulent carriers such a~
lS lactose, saccharose, sorbitol, mannitol, starches, e.g. potato starch or corn starch, or amylopectin, cellulose derivatives or gelatine, and may also include magnesium stearate or stearic acid as lubricants.

The compositions of the invention may be formulated æo as to provide quic~, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.

By using several layers of the active drug, separated by slowly dissolving coatings, sustained release tablets are obtained.
Another way of preparing sustained release tablets is to divide the dose of the active drug into granules with coatings of different thicknesæ and compress the granules into tablets together with the carrier substance.

The active substance can also be incorporated in slowly dissolving tablets made of e.g. fat and wax substances or evenly distributed in a tablet of an insoluble substance such as a physiologically inert plastic substance.

Liquid preparations for oral application may be in the form of elixirs, syrups or suspensions, e.g. solutions cont~ from SUE~s ~ JTE SHEET

W095/~27 16~9 ~1 PCT/SE94/00604 about 0.1% to 20~ by weight of the active substance, sugar and a mixture of ethanol, water, glycerol, propylene glycol and optionally aroma, saccharin and/or carboxymethylCellUlose as dispersing agents. The formulations can additionally include ~ 5 wetting agent, emulsifying and suspending agents, preserving agents and sweeteni~q agents.

For parenteral application by injection, preparations may comprise an aqueous solution of the active drug or a physiologically acceptable salt thereof, desirably in a concentration of 0.5-20% and optionally also a stabilizing agent and/or buffer substances in aqueous solution. Dosage units of the solution may advantageously be enclosed in ampoules.
There is limited knowledge of compounds that inhibit the adherence of Helicobacter pylori to mucosal surfaces such that the compounds are useful in the prevention or treatment of gastrointestinal disorders and ~i ~^A~95 caused or mediated by 20 Helicobacter pylori. Because of this limited knowledge, the dosage at which the active ingredients may be administered may vary within a wide range and will depend on various factors such as e.g. the severity 4f the infection, the age of the patient etc. and may have to be individually adjusted.
The phar~aceutical compositions of the subject invention preferably contain from about 1 mg to about SO g, more preferably from about lO mg to about 5 g per day of the active ingredient and may be divided into multiple doses.
The invention is further illustrated by the following, non-limi~ing examples.

SUB~ JTE SHEET

WO 95t00527 2i~ 36-Ph~o~ R Ph/~o,~ ~HO~C R

CH~ ~ 't OBn ~ CH3 ~ O; OBn ~ / g~ OBn B o OBn~ BnO

3 R=OMe 1 R=SEt 6R= ~
2 R=OMe 9R= O o~ O~N~
5R= ~~

8 R = O o - - o - N3 OH
\
J HNAc CH~ ~ OH
OH
HO

4 R = OMe 7R= --1~
10 R ~ ~~o~ ~\NH2 x CH3COOH

OH
HO~O~O o~O--NlN~ BSA
H H
_I HN~C
CH3~ 0 ~ OH
rOH

SUB~ )TE SHEEr WO 95/00527 215~961 PCT/SE94/00604 OH OBz - HO~--o R Ho~~5~ R
HO~ BzO~ OBn H3C~5_SEl 12 R = O Si(CH3)3 0 14 OBz =OJ~Ph 08n= OCH2Ph 08n HO
I OBn ¦ OH
H3C ~08n OB H3C ~OH OH

O~R H~R
~ c O NI~Ac .~

R ~ --Si(CH3)3 16 R ~ --Si(CH3)3 O O
OE~z _ O~Ph NHAc = HN~CH3 OBn ~ OCH2Ph SUB~ 1 1 1 ulTe SHEET

WO 95/OO!j27 PCT/SE94/00604 21 6~61 -38-OAC ~OH
PCO~VR HO~R
~o o~O

17 R = SEt 18 R = SEt OAC = O~CH3 OBn H3C~OBn ACO- ~Ro HO~

19 R5 SEt 21 R5 OMe 20 R OMe OBn= OCH2Ph 23 R- O~_ O
/~ NHAC = HN CH3 OBn = OCH2Ph OH
OAC ~ O~CH3 H3C !~OH

HO;;~,R
NHAC
22 R = OMe 24 R ~ ~

SUB~ I z I ~JTE SHEET' ~ -39- 21 6~96 ~

'' BnO~ \,.~SEt BnO~$\\vOR2 ORl OR1 R~_Ac 27 R~- Bz, R2s -CH2CH2N3 26 R~ = Bz 28 R~ s H, R2 ~ -CH2CH2N3 32 R~ - Bz, R2= -(CH2CH20)2CH2CH2N3 33 R~ = H, R2- -(CH2CH20)2CH2CH2N3 R~O OR
~Bn R10-~\ \v BnO
14 ~O~OR

29 R, =Bn, R2 - -CH2CH2N3 30 R~, H, R2- -CH2CH2NH2 57 Rl . H, R2 ~ -CH2CH2NHCOCH2 34 R1 - Bn, R2 - -(CH2CH20)2CH2CH2N3 35 R, =H, R2 - -(CH2CH20)2CH2CH2NH2 37 R~ ~ H, R2 s -(Cl~2CH20~2CH2CH2NHCOCHCH2 ~ o~H NHCN~HSA /HO~ O o~O--~NHCN~HSA

\r / ~ OH

. = .
:
- SUt~ 1 ITE SHEET~

%i~49~ -40-Ph~_ BnO~
HO NPhlh N3 BnO ~\ ~ R2 N3 o 44 Rl ~ AcR2 ~ NPh~h NPhth ~ N~ 45R~ =OH R2 5 NHAc Or R~ ~O RRo~_O
R10 ~\ - NHAc R2 CHa~ ~OR~
R~

46 Rl s Bn R2 s N3 R3 = CHPh 47 R~ = Bn R2 = NHCOCF~ R3 = CHPh 48 Rl = H R2 = NHCOCF3 R3 = H
49 Rl 5 H R2 = NHCOCHCH2 R3 = H

SUB~ JTE SHEEr wo gsl00s27 -41- 216q9 BnO OBn OR3 ~_0 R40~_0 BnO ~ OR~ R2 N3 45 R~ = H R2 = NHAc R3, R4 = CHPh 51 R1=H R2=NHAc R4=H R3 =OBn R~O
¦ OR~
CH35~oR, 1 ~ 0--~

R10 ~0\ NHAc R2 CH3~ OR, R~O

52 R, = Bn R2 = N3 R4 ~ Bn 53 R~. Bn R2= NHCOCF3 R4 =Bn 54 R~ z H R2 = NHCOCF3 R4 = H
55 R~ _H R2- NHCOCH2 R4 ~ H

SUB~ 111 L)TE SHEEr wo gsl00s27 2~G 4~6~ -.H

. ~ H~
CH3~H
OH
OH
Fuca1 -2Gal~1 -3(Fuc1 -4)GlcNAc~1 -3Gal~1 -4Glc~ -R

40 Rl = NH2 41 Rl = NHCOCHCH2 42 R2 = Fuca1-2Gal~1~3(Fuc~1~4)GlcNAc~1~3Gal~1~4Glc~l -NH H2N--C~
38 R2 = Fuca1-2Gal~1-O(CH2CH20)2CH2CH2NH R2~C---58 R2 = Fuc1-2Gal,B1-CH2CH2NH H2N--C--50 R2 = Fuc~1-2Gal~1-3GlcNAc~1-CH2CH2NH
56 R2 s Fuc1-ZGal~1-3(Fuc~1-4)GlcNAc~1-CH2CH2NH

SUB~ 111 ~JTE SHEET

woss/~s27 ~ pcTlsEs4loo6o4 General metho~o lH and 13C NMR spectra in examples 1 to 6 were recorded on a Varian Gemini 300 spectrometer and on a Varian Unity 400 MHz

- 5 spectrometer. In examples 1 to 6 the following reference signals were used: CHC13, ~ 7.25 (lH in CDCl3); CHCl3, ~ 77.9 (13C in CDCl3); (CH3)2CO, ~ 2.24 or CHD2OH ~ 3.31 (1H in D2O);
tCH3)2CO, ~ 33.19 or CHD2OH, ~ 51.89 (13C in D2O); CHD2OH, 3.31 (lH in CD30D). lH andl3C NMR spectra in all other examples were recorded at 25C in CDC13 (using tetramethylsilane as internal st~nAArd for lH, CDCl3 ~ 77.0 for 3C) and in D2O (HDO ~ 4.765 for lH, us~ng aceton ~ 30.0 as internal st~n~rd for 13C). NMR spectra recorded for all compounds were in agreement with the structures postulated and only selected data are reported. Masspectra to determine the degree of substitution of carbohydrate component vs. protein were performed on a VG TOFSPEC linear time of flight masspectrometer. Fab-NS was run on a Nermag 1010L, with an Iontech FAB gun and a matrix of thioglycerol. Optical rotations were measured using a Perkin Elmer 241 polarimeter. Thin layer chromatography (TLC) was performed on Nerck DC-Fertigplatten (Kiselgel 60 F254 0.25 mm) and spots were visualized by W or by spraying with 10% sulphuric acid followed by charring at elevated temperature, or by spraying with phospohomolybdic acid or ninhydrin in n-butanol (0.5%). Silica gel 60 (40-63 Am) and Amicon Matrex2 Silica Si 0.35-0.70 m was used for column chromatography.
Separations were also performed on a Chromatotron rotary TLC
using 1--2 mm layers of Silica Gel 60 PF254 with gipsum. All Biogel P-2 column were eluated with 1% n-buthanol in deionized water if not otherwise stated.

~ethyl ~-acet~mi~o-2-deoxy-3-O-a-L-fucG~ ~osyl-~-D-glu~o~y~oside (~ ) SUB~ TE SHEET

W095/00~7 2 1 6 ~`9 6 1 PCT/SE94/00604 (i) Methyl 4,6-0-benzylidene-3-0-(tri-0-benzyl-~-L-fucopyranosyl)-2-deoxy-2-phthalimido-~-D-glucopyranoside (2) Trifluoromethanesulfonic acid (2 ~1, 0.023 mmol) was added to a stirred mixture of ethyl 3-o-(tri-0-benzyl-~-L-fucopyranosyl)-4~6-o-benzylidene-2-deoxy-2-phthalimido-l-thio-~-D-glucopyranoside (1) (100 mg, 0.117 mmol), (prepared according to H. Lonn, Carbohydr. Res. 139 (1985), 105-113) methanol (7 ~1, 0.175 mmol), N-iodosuccinimide (40 mg, 0.175 mmol) and ground molecular sieves (100 mg, 3A) in dichloromethane-diethyl ether (3 ml, 2:1) at -30C. After 45 min the reaction mixture was filtered through a layer of Celite into an aqueous solution of sodium hydrogen carbonate and sodium bisulphite. The organic layer was separated, washed with aqueous sodium chloride, and concentrated. Column chromatography (toluene-ethyl acetate, 20:1) of the residue gave amorphous (2) (93 mg, 97 %), ~a~D -16.2 (c 1.0, CHCl3).

1H NMR data (CDC13, ~): 7.80 to 7.00 (24H, benzyl and phthaloyl), 5.29 (d, lH, J 8.6 Hz, H-l), 4.84 to 4.25 (SH, CH2Ph), 4.84 (bs, lH, H-l'), 4.66 (dd, lH, J 8.5 and 10.3 Hz, H-3), 4.48 to 4.43 ~m, lH, H-3'), 4.35 (dd, lH, J 8.6 and 10.3 Hz, H-2), 4.08 (bdd, lH, J 6.4 and 13.0 Hz, H-5'), 3.91 to 3.81 (2H), 3.78 to 3.66 (4H), 3.51 to 3.47 (lH), 3.48 (s, 3H, OCH3), 0.90 (d, 3H, CH3).

3C NMR data (CDCl3, ~): 168.0 (CO), 138.8 to 123.0 (benzyl), 101.1 (CHPh), 99.7 (C-l'), 99.4 (C-l), 82.1, 79.5, 78.0, 75.7, 75.5, 74.6, 73.0, 72.5, 68.6, 67.2 (C-5'), 66.1, 56.9 (OCH3), 55.5 (C-2), 16.3 (CH3).

(ii) Methyl 2-acetamido-3-0-(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-4~6-o-benzylidene-2-deoxy-~-D-glucopyranoside 35 (3) A solu~ion of (2) (1.13 g, 1.36 mmol) and hydrazine hydrate (3.3 ml, 68 mmol) in agueous 95% ethanol was boiled under SUB;~ JTE SHEEI-W095/~27 1 6~.9 61 PCTISE94/00604 reflux for 20 h, ccoled, and concentrated. The residue was acetylated with acetic anhydride-pyridine (50 ml, 1:1) overnight. The solution was concentrated, and the residue was subjected to column chromatography (heptane-ethyl acetate, 1:1) S to give crude (3) ~.hich was used directly in the next step.

H NMR data (CDC13, ~): 7.50 to 7.25 (20H, benzyl), 5.71 (d, lH, J 7.4, NH), 5.52 (s, lH, CH2Ph), 5.09 (d, lH, H-1'), 4.85 to 4.58 (6H, CH2Ph), 4.82 (d, lH, H-l), 4.37 )dd, lH, J 4.6 and 10.4 Hz, H-6), 4-.28 (bt, lH, H-3), 4.12 to 4.05 (2H, H-2' and H-5'), 3.95 (dd, lH, J 2.6 and 10.2 Hz, H-3'), 3.78 (bt, lH, H-6), 3.63 (bs, lH, H-4'=, 3.60 (bt, lH, H-4), 3.53 (m, lH, H-5), 3.48 (s, 3H, OCH3), 3.42 (ddd, lH, J 7.2, 8.2 and 9.5 Hz, H-2), 1.67 (s, 3H, NHAc), 0.84 (d, 3H, CH3).
13C NMR data (CDC13, ~): 170.6 (C0), 138.6 to 126.2 (benzyl), 101.8 (~-1), 101.6 (CHPh), 98.4 (C-l'), 80.8 (C-4), 79.8 (C-3'), 77.6 (C-4'), 77.0 (C-2' or C-5'), 75.1 (C-3), 74.9 (CH2Ph), 72.5 (CH2Ph), 68.8 (C-6), 66.9 (C-2' or C-5'), 66.2 (C-5), 58.1 (C-2), 57.0 (OCH3), 23.2 (NHAc), 16.3 (CH3).

(~ii) Methyl 2-acetamido-2-deoxy-3-O-~-L-fucopyranosyl-~-D-glucopyranoside (4) 2S A solution of crude (3) (1.05 g) in acetic acid-ethyl acetate-water (9:5:1, 120 ml) was hydrogenolysed at 200 kPa over 10%
Pd/C (1 g) over night. The mixture was filtered through a layer of Celite and conc~ntrated. Column chromatography (chloroform-methanol-water, 65:35:6) of the residue gave amorphous ~ (469 mg, 90% calculated from (2), t~D -116.0 (C 1.0, water).

lH NMR data (D20, acetone ref., ~): 4.99 (d, lH, J 4.0 Hz, H-l'), ~.46 (d, lH, J 8.7 Hz, H-l), 4.33 (bdd, lH, H-5'), 3.98 to 3.45 (9H), 3.51 (s, 3H, OCH3), 2.03 (s, 3H, NHAc), 1.17 )d, 3H, CH3).

SUB~ I 11 ~JT~ SHEET

WOg5/~7 2 1 6 ~ 9 6 1 PCT/SE94/00604 ~

13C NMR data (D20, acetone ref., ~3: 177.6 (C0), 104.7 (C-l), 102.9 (C-l'), 83.5, 78.8, 74.7, 72.5, 71.6, 70.9, 69.9, 63.7, 60.0, 59.1 (C-2), 25.2 (NHAc), 18.1 (CH3).

~SAMP~E 2 3,3-D~Qthylbutyl 2-acet~ido-2-~eoxy-3-0-~-L-fUcopyr~no~yl-~-D-glucG~.anos~- (7) (i) 3,3-Dimethylbutyl 3-0-(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-4~6-o-benzylidene-2-deoxy-2-phthalimido-~-D-gluco~Lanoside (5) Trifluoromethanesulfonic acid (30 ~1, 0.35 mmol) was added to a stirred mixture of (1), 3,3-dimethyl-butan-1-ol (317 ~1, 2.62 mmol~, N-iodosuccinimide (602 mg, 2.62 mmol), and ground molecular sieves (1.5 g, 3A) in dichloromethane-diethyl ether (2:1, 45 ml) at -30C. After 45 min the reaction mixture was filtered through a layer of Celite into an aqueous solution of sodium hydrogen carbonate and sodium bisulphite. The organic layer was separated, washed with aqueous sodium chloride, and concentrated. Column chromatography (heptane-ethyl acetate,

6:1) of the residue gave amorphous (5) (1.42 g, 90~), t~]D

-22.2 (c 1.0, CHC13).
lH NMR data (CDC13, ~): 7.80 to 7.0 (24 H, benzyl and phthaloyl), 5.57 (s, lH, CHPh), 5.35 (d, lH, J 8.5 Hz, H-1), 4.84 (bs, lH, H-l'), 4.83 to 4.24 (5H, CH2Ph), 4.65 (dd, lH, J
8.3 and 10 3 Hz, H-3), 4.34 (dd, lH, J 8.5 and 10.4 Hz, H-2), 4.07 (dd, lH, J 5.5 and 10.4 Hz, H-5'), 3.96 to 3.66 (7 H, inter alia OCH2), 3.53 to 3.45 (2H, inter alia OCH2), 1.46 to 1.29 (m, 2H, CH2C(CH3)3), 0.88 (d, 3H, J 6.4 Hz, CH3), 0.73 (s, 9H, CH2C(CH3)3)-13C NMR data (CDC13, ~): 168.0 (C0), 138.8 to 123.0 (benzyl and phthaloyl), 101.1 (CHPh), 99.4 (C-l'), 98.9 (C-l), 82.1, 79.5, 76.0, 75.7 (C-3), 75.6, 74.6, 73.0, 72.6, 68.7, 67.3 (OCH2), SUB:~ 111 LITE SHEET

woss/005~7 216~961 PCT/SE94/00604 67.2 (C-5), 66.2, 55.8 (C-2), 42.4 (CH2C(CH3)3), 30.8 (CH2C(C~3)3), 29.4 (CH2C(CH3)3), 16-3 (CH3).

(ii) 3,.~-Dimethylbutyl 3-0-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-2-acetamido-4~6-o-benzylidene-2-deoxy-~-D-glucopyranoside (6) A solution of (5) (1.42 g, 1.58 mmol) and hydrazine hydrate (3.9 ml, 79 mmol) in aqueous 90% ethanol (100 ml) was boiled under reflux for 20 h, cooled, and concentrated. The residue was acetylated with acetic anhydride-pyridine (50 ml, 1:1) overnight. The solution was concentrated. Column chromatography (heptane-ethyl acetate, 3:1, containing 1% methanol) of the residue qave amorphous (6) (1.16 g, 90%), t~]D ~74-7 (c 1.0, CHC13).

H NMR data (CDC13, ~): 7.50 to 7.20 (20 H, benzyl), 5.63 (d, lH, J 7O3 Hz, NH), 5.51 (s, lH, CH2Ph), 5.07 (d, lH, J 3.1 Hz, H-l'). 4.93 to 4.57 (6H, CH2Ph), 4.92 (d, lH, H-1), 4.39 to 4.29 (2I~, H-6 and H-3), 4.11 to 4.04 (2H, H-2'and H-5'), 3.98 to 3.85 (2H, H-3'and OCH2), 3.77 (bt, lH, H-6), 3.61 (bs, lH, H-4'), 3.55 (bt, lH, H-4), 3.59 to 3.44 (2H, H-5 and OCH2), 3.33 (bdd, lH, H-2), 1.63 (s, 3H, OAc), 1.56 to 1.40 (m, 2H, CH2C(CH3)3), 0.89 (s, 9H, CH2C(CH3)3), 0.82 (d, 3H, CH3).
3C NMR data (CDC13, ~): 170.4 (Co), 138.6 to 126.0 (benzyl), 101.6 (CHPh), 100.7 (C-l), 98.1 (C-l'), 80.9 (C-4), 79.8 (C-3'), 77.6 (C-4'), 77.0 (C-2'or C-5'), 74.9 (C-3), 74.8 (CH2Ph) r 74.0 (CH2Ph), 72.5 (CH2Ph), 68.9 (OCH2 or H-6), 67.5 (OCH2 or H-6), 66.8 (C-5' or C-2'), 66.2 (C-5), 58.6 (C-2), 42-7 (CII2C(CH3)3)~ 29-7 (CH2C(CH3)3), 29.6 (C(CH3)3), 23.2 (NHAc), 16.2 (CH3).

(iii) 3l3-Dimethylbutyl 2-acetamido-2-deoxy-3-O-~-L-fUcopyranosyl-~-D-glu~G~y ~noside (7) A solution of the compound (6) (1.08 g, 1.33 mmol) in acetic acid:ethyl acetate:water, 9:5:1 (120mL) was hydrogenolysed at SU~ JTE SHEEr wo gs/00s27 2 1 6 ~ 9 6 ~ - 48- PCT/SE94/00604 200 kPa over 10% Palladium on charcoal (Pd/C) (1 g) over night.
The mixture was filtered through a layer of Celite and concentrated. Column chromatography (chloroform-methanol-water, 100:30:3) of the residue gave amorphous (7) (566 mg, 94%), t~]D
S -109-7 (c 1.0, water).

H NMR data (D20, acetone ref., ~): 4.99 (d, lH, H-l'), ~.84 (g, lH, CHPh), 4.34 (bdd, lH, H-5'), 4.02 to 3.43 (llH), 2.01 (s, 3H, NHAc), 1.57 ti 1.41 (m, 2H, CH2C(CH3)3), 1.17 (d 3H, CH3), 0.90 (8, 9H, C(CH3)3)-3C NMR data (D20, acetone ref., ~): 177.3 (C0), 103.6 (C-l), 102.8 (C-l'), 83.6, 78.8, 74.8, 72.5, 71.6, 70.9, 69.8, 63.7, 58.1 (C-2), 44.9 (CHC(CH3)3), 31.9 (C(CH3)3), 31-9 )C(CH3)3), 25.2 (NHAc), 18.1 (CH3).

~XAM~g 3 ~Ucal-3GlcNAc~1-0-~p-~r~ 1-B8A-con~ugate (11) (i) 8-Azido-3,6-dioxaoctyl 3-0-(2,3,4-tri-0-benzyl-~-L-~u~y.anosyl)-4,6-0-benzylidene-2-deoxy-2-phthalimido-~-D-glucopyranoside (8) Trifluoromethanesulfonic acid ~24 ~1, 0.27 mmol) was added to a stirred mixture of (1) (1.15 g, 1.34 mmol), 8-azido-3,6-dioxaoctan-1-ol (352 ~1, 2.01 mmol) (prepared according to P.
H. Amvam-Zollo and P. Sina~, Carbohydr. Res. 150 (1986), 199-212), N-iodosuccinimide (461 mg, 2.01 mmol) and ground molecular sieves (1.15 g, 3A) in dichloromethane-diethyl ether (30 ml, 2:1) at -30C. After 1 h the reaction mixture was filtered through a layer of Celite into a aqueous solution of sodium hydrogen car~onate and sodium bisulphite. The organic layer was separated, washed with aqueous sodium chloride, and concentrated. Column chromatography (heptane-ethyl acetate, 2:1) of the residue gave amorphous (8) (1.03 g, 79%), t~]D
-21-7 (c 1.0, CHC13).

SUBS I ~ I ~JTE SHEET

W095/~527 21 B~ 9 61 pcTlsæ94loo6o4 lH NMR data (CDC13~ 7.80 to 7.05 (24H, Bzl, Phth), 5.59 (s, lH, CHPh), 5.44 (d, lH, J 8.6 Hz, H-l), 4.83 (bs, lH, H-l'), 4.83 to 4.24 (SH, CH2Ph), 4.65 (dd, lH, J 8.5 and 10.3 Hz, H-3), 4.45 to 4.41 (lH, H-3'), 4.39 (dd, lH, J 8.6 and 10.3 Hz, ~ 5 H-2), 4.09 (dd, lH, J 6.4 and 12.6 Hz, H-5'), 3.99 to 3.83 (3H, inter alia OCH2), 3.81 to 3.68 (5H, inter alia OCH2), 3.61 to 3.30 (llH, inter alia OCH2 and CH2N3), 0.90 (d, 3H, J 6.4 Hz, CH3)-13C NMR data (CDC13, ~): 169.0 (C0), 138.9 to 123.1 (Bzl, Phth), 101.1 (CH2Ph), 99.4 (C-l'), 98.9 (C-l), 82.1, 79.6, 78.0, 75.6, 75.5, 74.7, 73.1, 72.6, 70.5, 70.4, 70.1, 69.9, 69.~, 68.7, 67.2, 66.2, 55.7 (C-2), 50.6 (CH2N3), 16.4 (CH3).

(ii) 8 Azido-3,6-dioxaoctyl 2-acetamido-3-0-(2,3,4-tri-0-benzyl ~-L-fucopyranosyl)-4,6-O-benzylidene-2-deoxy-~-D-glucopyranoside (9) A solution of (8) (633 mg, 0.65 mmol) and hydrazine hydrate (1.6 ml, 33 mmol) in aqueous 90% ethanol (45 ml) was boiled under reflux for 24 h, cooled, and concentrated. The residue was acetylated with acetic anhydride-pyridine (50 ml, 1:1) overnight. The solution was concentrated. Column chromatography (chloroform-acetone, 9:1) and re-chromatography (ethyl acetate-heptane, 3:1) of the residue gave amorphous (9) (440 mg, 77~), t~D -72.6 (c 1.0, CHC13).

H NMR data (CDC13, ~): 7.50 to 7.25 (20H, benzyl), 7.92 (d, lH, J S.9 Hz, NH), 5.51 (s, lH, CHPh), 5.16 (d, lH, J 3.5 Hz, H-l ~, 4.93 (d, lH, J 7.7, H-l), 4.95 to 4,56 (6H, CH2Ph), 4.34 (dd, lH, J 4.9 and 10,4 Hz, H-6), 4.26 (bt, lH, H-3), 4.12 (bdd, lH, H-5'), 4.06 tdd, lH, J 3.5 and 10.1 Hz, H-2'), 3.95 (dd, lH, J 2.7 and 10.1 Hz, H-3'), 3.90 (bt, lH, H-6), 3.81 to 3.45 (14H, inter alia OCH2), 3.40 (m, 2H, CH2N3), 1.74 (s, 3H, NHAc), 0.84 (d, 3H, J 6.4 Hz, CH3).

3C NMR data (CDC13, ~): 170.4 (C0), 138.7 to 126.1 (benzyl), 101.5 (CHPh), 101.2 (C-l), 97.8 (C-l'), 80.6 (C-4), 79.6 SIJB~ JT~ SHEET

wos5/~27 pcTlsæ94loo6o4-21~49~1 -so~
(C-3'), 77.7 (C-4 ~ 76.7 (C-2~ or C-5~), 74.9 (C-3), 74.6 (CH2Ph), 73.7 (CH2Ph), 72.2 (CH2Ph), 70.6 (CH20), 70.6 (CH2O), 70.4 (CH20), 69.9 (CH2O), 68.8 (CH2O), 68.8 (H-6), 66.7 (C-2' or C-5'), 66.2 (C-5), 57.8 (C-2), 50.6 (CH2N3), 23.2 (NHAc), 16.2 (CH3)-(ii~) 8-Amino-3,6-dioxaoctyl 2-acetamido-2-deoxy-3-O-~-L-fucopyranosyl-~-D-glucopyranoside acetic acid salt (10) A solution of (9) (57 mg, 0.065 mmol) in acetic acid-water (9:1, 30 ml) was hydrogenolysed at 200 kPa over 10% Pd/C (100 ~ mg) over night. The mixture was filtered through a layer of Celite and concentratéd. The residue was first subjected to column chromatography on silica gel (chloroform-methanol-water, 4:4:1) and then on A12O3 ~Merck, basic, 0.063-0.200 mm, chloroform-methanol-water, 4:4:1) to give amorphous (10) (18 mg, 51~), t~D -70.6 (c 0.2, water).

1 H NMR data (D2O, acetone ref., ~): 4.98 (d, lH, J 4.0 Hz, H-l'). 4.53 (d, lH, J 8.6 Hz, H-l). 4.32 (bdd, lH, H-5'), 4.05 to 3.42 (19H), 3.19 (m, 2H, CH2NH2), 2.01 (s, 3H, NHAc), 1.88 (CH3COOH), 1.14 (d, 3H, J 6.6 HZ, CR3).

13C NMR data (DzO, acetone ref., ~): 184.2 (CH3COOH), 103.8 (C-l), 102.8 (C-l'), 83.3, 78.8, 74.8, 72.6, 72.5 72.4, 72.0, 71.5, 70.9, 69.8, 69.4, 63.7, 58.1 (C-2), 42.0 (CH2NH2), 26.3 (CH3COOH), 25.2 (NHAc), 18.1 (CH3).

tiV) Fuc~1-3GlcNAc~l-o-Spacer l-BSA-conjugate (11) Thiophosgene (67 ~1, 0.856 mmol) in acetone (6 ml) was added dropwise to an ice-cold solution of (10) (120 mg, 0.214 mmol) in water-ethanol-0.1 N phosphate buffer pH 7 (1:1:1, 30 ml).
The pH was kept at 6-7 with aqueous sodium hydroxide (1 M) during the reaction. After 20 min the mixture was extracted with diethyl ether (30 ml), concentrated to a volume of 10 ml, and added to a solution of bovine serum albumin (695 mg, 10.7 mmol) in a~ueous sodium hydrogen carbonate (15 ml, 0.1 M, pH

SUB~ ~ .JTE SHEET

W095/~27 21 6 ~ 9 61 PCT/SE94/00604 9.3). During the addition, pH was adjusted to g with aqueous sodium hydroxide (1 M). After 24 h the reaction mixture was desalted by ultrafiltration (Filtron, omegacell 150, 10 K) and freeze-dried to give (11) (672 mg). The degree of substitution S was determined by sugar analysis (see M. A. Jermyn, Anal. Chem.
68 (1975), 332-335) to 15-18 mol disaccharide/mol protein.

EgAMPL~ 4 2-Trimethylgilylethyl 2-acot~mi~o-2-deoxy-~-O-~-L-fuco-pyr~nosyl-~-D-glucGy~.~no~ide ~6) (i) Trimethylsilylethyl 3,6-di-O-benzoyl-2-deoxy-2-phthalimido-~-D-glucopyranoside (13) 2-Trimethylsilylethyl-2-deoxy-2-phthalimido-~-D-glucopyranoside (12) (1.64 g, 4.0 mmol) (prepared as described by K. Jansson, S. Ahlfors, T. Frejd, J. Rilhberg, G. Magnusson, J. Dahmén, G.
Noori, and K. Stenvall, J. Org. Chem. 53 (1988), 5629-5647), was ~ clved in pyridine-dichloromethane (3:1, 24 ml) and cooled to -45C. A mixture of benzoyl chloride (1060 ml, 9.1 mmol) and pyridine (900 ml) was added during 30 minutes. The reaction was completed after 3 hours and methanol (40 ml) was added. The solvents were evaporated and the residue was co-evaporated with toluene 3 times. The residue was chromatographed (SiO2, heptane/ethyl acetate 2~ to give pure (13) (2.38 g, 96%). ~D20 l69.4 (c 1.2, CHCl3).

lH NMR data (CDC13, ~): d 7.3-8.2 (m, 14H, 2 O-benzyl, N-phthalamoyl), 5.88 (dd, lH, J 7.1, 8.3 Hz, H-3~, s.46 td, lH, J 8.5 Hz, H-l), 4.79 (dABq, lH, J 4.2, 12.4 Hz, H-6), 4.67 (dABq, lH, J 1.9, 11.7 Hz, H-6), 4.45 (dd, lH, J 8.4, 10.8 Hz, H-2), 3.8-4.1 (m, 3H, H-4, H-5, OCH2CH2), 3.57 (dt, lH, J 7.2, 9.7 Hz, OCH2CH2), 0.7-1.0 (m, 2H, CH2Si), -0.15 (s, 6H, SiMe3).
(ii) 2-Trimethylsilylethyl 3,6-di-O-benzoyl-4-O-(2,3,4-tri-o-benzyl-~-L-fucopyranosyl)-2-deoxy-2-phthalimido-~-D-glucopyranoside (15) .~
SUB~ TE SHEEr Wog~/00527 216 ~ 9 G 1 PCT/SE94/00604 Compound (13) was dissolved in dichloromethane-N,N-dimethylformamide (8 ml, 5:3) and tetrabutylammonium bromide (664 mg, 2.06 mmol) and molecular sieves (4 A, 4 g, activated) was added. To a solution of thioethyl 2,3,4-tri-O-benzyl-1-thio-~-L-fucopyranoside (14) (986 mg, 2.06 mmol) (prepared according to H. L~nn, Carbohydr. Res. 139 (1985), 105-113) in dichloromethane (8 ml) was added bromine (122 ml, 2.37 mmol) in dtchloromethane (2 ml). After 15 min stirring, cyclohexene (distilled) was added dropwise until the bromine colour disappeared. This solution was then added to the mixture above containing compound (13) and stirred for 48 h. The mixture was then filtered through Celite, the solvents were evaporated and the residue was co-concentrated with toluene three times.
Column chromatography of the residue (heptane/ethyl acetate, S:l~ 1:1) gave (15) (896 mg, 85%), t~]D20 + 14.6 (c 1.2, CHC13).

lH NMR data (CDC13, ~): 5.44 (d, lH, J 8.5 Hz, H-l), 4.80 (d, lH, J 3.6 Hz, H-1').
(iii) 2-Trimethylsilylethyl 2-acetamido-2-deoxy-4-O-(~-L-fuco-pyranosyl)-~-D-glucopyranoside (16) Compound (15) (760 mg, 0.74 mmol) was dissolved in methanol (7 ml) and sodium methoxide (220 ml, 2 M in methanol) was added.
The solution was stirred over night at room temperature and then neutralized with Amberlite IR-120(H). Filtration and evaporation of the solvents gave a syrup. The syrup was dissolved in acetic acid (15 ml) and 10% Pd/C (860 mg) was ~0 added. After l.S h hydrogenolysis (lO0 kPa), the mixture was filtered and the solvents evaporated. The resulting syrup was dissolved in ethanol (18 ml) and hydrazine hydrate was added.
The solution was refluxed for 3 h. Evaporation of the solvents and co-evaporation with ethanol S times gave a syrup that was dissolved in methanol-water mixture (5:1, 60 ml). Acetic anhydride (5 ml) was added and the solution was stirred for 1.5 h. The solvents were evaporated. Column chromatography (sio2~

SUB~ 111 ~TE SHEET

woss/oos~l 1 ~ 9 61 PCT/SE94/00604 dichloromethane/methanol, 5:1) gave (16) (lOo mg, 29%), t~]D20 -113.5 (c 0.7, H20).

H NMR data (D20, ~): d 4.93 ~d, lH, J 3.66 Hz, H-1'), 4.52 (d, ~ 5 lH, J 8.06 Hz, H-1).

EXAMPLE S

Mathyl 2-acet~mi~o-2-deoYy-6-O-~-~-fu~G~y~nosy~ D-glucG~y~nosi~ ~22) (i) Ethyl 2-deoxy-2-phthalimido-1-thio-~-D-glUCopyranoside (18) Ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-~-D-glucopyranoside (17) (5.79 g, 12 mmol) (prepared according to H. Lonn, Car~ohy~r. Res. (1985) 139, 105-113) was dissolved in methanol (250 ml) and methanolic sodium methoxide (0.2 M, 2.5 ml) was added. The mixture was stirred for 15 h. Neutralization with acidic cation exchange resin (Bio-Rad AG~ 50W-X8), filtration, evaporation and crystallization from water ~ave (18) (3083 g, 89%), m.p. approx. 96C; m.p. after recrystallization 159-161C; ta3D22 +9.8 (c 0.9, methanol).

lH NMR data (CD30D, CHD2OD ref., ~) d: 7.91-7.79 t5H), 5.32 (d, lH, J 10.5 Hz, H-l), 4.28 (dd, lH, J lo and 8 Hz, H-3), 4.05 (t, lH, J 10.5 Hz, H-2), 3.93 (dd, lH, J 12 and 2 Hz, H-6), 3.73 (dd, lH, J 12 and 5.5 Hz, H-6), 3.46 (ddd, lH, J 10, 5.5 and 2 Hz, H-5), 3.40 (dd, lH, J 10 and 8 Hz, H-4), 2.74 (dg, lH, J 12.5 and 7.5 Hz, SCH), 2.63 (dq, lH, J 12.5 and 7.5 Hz, SCH) ancl 1.17 (t, 3H, J 7.5 Hz, CH3CH2).

(ii) Ethyl 3,4-di-O-acetyl-6-O-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-2-deoxy 2-phthalimido-1-thio-~-D-glucopYranOSide (19) 3s Bromine (0.485 ml, 9.4 mmol) was added to a solution of ethyl 2,3,4-tri-O-benzyl-l-thio-~-L-fucopyranoside (14) (4.5 g, 9.4 mmol) in dichloromethane (70 ml) at 0C. The mixture was SUB~ JT~ SHEET

W095l~7 ~ 21~9GI PCT/SE94/0060 ~

stirred for 35 min and was then evaporated twice with benzene.
Cyclohexene (0.5 ml) was added and the mixture was again evaporated with benzene. The residue was dissolved in dichloromethane (2S ml) and then added during 1 h, to a stirred mixture of compound (18) (3.32 g, 9.4 mmol), powdered molecular --sieves (20 g, 4 A) and tetraethylammonium bromide (3.5 g) in dimethylformamide (75 ml). The reaction mixture was stirred for 2 h at 0C, then for 2 h at room temperature followed by filtering through Celite. The filtrate was partitioned between dichloromethane and saturated aqueous sodium hydrogen carbonate. The aqueous phase was extracted with dichloromethane and the combined organic phases were washed with water, and concentrated. The residue was chromatographed (ethyl acetate-heptane; 1:1-3:1) to give a crude product, which was O-acetylated by stirring in acetic anhydride (50 ml) and pyridine (75 ml) for 17 h at room temperature. Evaporation with toluene and chromatography (ethyl acetate-heptane; 1:2-1:3) gave tl9) (3.3 g, 40S), t~]D22 -4.6 (c 1.4, chloroform).

lH-NMR data (CHC13, ~): 7.90-7.83 (2H), 7.79-7.71 (2H),

7.45-7.24 (lSH), 5.83 (dd, lH, J 10 and 9.5 Hz, H-3), 5.43 (d, lH, J 10.5 Hz, H-1), 5.09 (dd, lH, J 10 and 9.5 Hz, H-4), 4.99 and 4.67 (2H, AB-system, J 11.5 Hz, benzylic H), 4.97 (d, lH, J
3.5 Hz, 8-1'), 4.89 and 4.77 (2H, AB-system, J 12 Hz, benzylic H), 4.79 and 4.73 (2H, AB-system, J 12 Hz, benzylic H), 4.39 (t, lH, J 10.5 Hz), 4.06 (dd, lH, J 10 and 3.5 Hz), 3.97-3.87 (3H), 3.76 (dd, lH, J 12 and 6 Hz), 3.70- 3.62 (2H), 2.67 (dq, lH, J 12 and 7.5 Hz, SCH), 2.56 (dq, lH, J 12 and 7.5 Hz, SCH), 1.98 (s, 3H, CH3C0), 1.87 (s, 3H, CH3C0), 1.15 (t, 3H, J 7.5 Hz, C~3CH2), 1.13 (d, 3H, J 7.5 Hz, FUC-CH3).

Calc. for C47HslN012S: C 66.1; H 6.02; N 1.64; S 3.75. Found: C
66.4; H 6.1; N 1.55; S 3.25.

(iii) Methyl 3,4-di-O-acetyl-6-O-(2,3,4-tri--O-benzyl-~-L-fucopyranosyl)-2-deoxy-2-phthalimido-~-D-glucopyr anoside (20j ..
SUt~ JTE SHEET

wos5t~527 21 6~9~1 pcT/sæ94loo6o4 To a mixture of (19.) (853 mg, 1 mmol), methanol (0.102 ml, 2.5 mmol), N-iodosuccinimide (344 mg, 1.52 mg) and ground molecular sieves (0.9 g, 4 A) in dichloromethane-diethyl ether (2:1, 25 ml) at -30C, was added trifluoromethanesulphonic acid (0.030 - 5 ml, 0.3 mmol). After 2.5 h. the reaction mixture was filtered through Celite into an aqueous solution of sodium hydrogen carbona~e and aqueous sodium bisulphite. The organic phase was separated and washed with saturated aqueous sodium chloride, and concentrated. Chromatography (ethyl acetate- heptane; 2:3) of the residue gave (20) (778 mg, 94S), ta]D22 -2.8 (c 1.1, chloroform).

lH NMR data (CHC13, ~): 7.90-7.82 (2H), 7.78-7.70 (2H), 7.45-7.24 (15H), 5.79 (dd, lH, J 11 and 9 Hz, H-3), 5.26 (d, lH, J 805 Hz, H-1), 5.08 (dd, lH, J 10 and 9 Hz, H-4), 4.99 and 4.67 (A~-system, 2H, J 11.5 Hz, benzylic H), 4.96 (d, 1~, J 3.5 Hz, H-l~), 4.88 and 4.77 (AB-system, 2H, J 12 Hz, benzylic H), 4.81 and 4.69 (AB system, 2H, J~12 Hz, benzylic H), 4.28 (dd, lH, J 1~ and 8.5 Hz, H-2), 4.07 (dd, lH, J 10 and 3.5 Hz), 3.99-3.85 (3H), 3.77 (dd, lH, J 12 and 6 Hz), 3.71-3.64 (2H), 3.34 (s~ 3H, CH30), 2.00 (s, 3H, CH3CO), 1.86 (s, 3H, CH3CO), 1.14 (dr 3H, J 6.5 Hz, FUC-CH3).

Calc. for C46H49N0l3: C 67.06; H 5.99; N 1.70. Found: C 67.0; H
6.1; N 1.65.

(iv) Methyl 2-acetamido-6-0-(2,3,4-tri-0-benzyl-~-L--fucopyranosyl)-2-deoxy-~-D-glucopyranoside (21) Compound( 20) was deacetylated in methanolic sodium methoxide (9.5 mM, 31.5 ml) for 1.5 h. Neutralization with acidic cation eYchAnge resin (Bio-Rad AG~ 50W-X8), filtration and concentration gave a residue which was dissolved in methanol (20 ml). Hydrazine monohydrate (0.8 ml) was added and the mixture was heated under reflux for 4 h and then cooled to 10C. Water (15 ml) and acetic anhydride (5 ml) were added and the reaction mixture was stirred at room temperature. After 20 min a white precipitate was obtained. Addition of methanol (10 SUB~ 1-1 ~JTE SHEET

WO 95/00527 PCT/SE~,'C0 60~
2~9~_s6_ ~
ml) facilitated stirring. After additional 2.5 h, pyridine (2 ml) was added which resulted in a clear solution. The mixture was then stirred for 30 min. The methanol was evaporated and the aqueous residue was extracted with dichloromethane. The organic phase was washed with 1 M HCl and saturated aqueous sodium hydrogen carbonate, and concentrated. Chromatography (ethyl acetate-methanol, 10:1) of the residue gave (21) (435 mg, 77~). An analytical sample was crystallized from ethanol, m.p. 224-226OC (d), t~]D22 -75.4 (c 0.9, CHC13).
H NMR data (CDC13-CD30D, 3:1, CHD20D ref., ~) d: 7.41-7.20 tl5H), 4.91 and 4.61 (AB-system, 2H, J 11.5 Hz, benzylic H), 4.80 and 4.70 (AB-system, 2H, J 11.5 Hz, benzylic H), 4.78 (d, lH, J 3 Hz, ~-1'), 4.75 (s, 2H, benzylic H), 4.24 (d, lH, J 8.5 Hz, H-1), 4.09-3.97 (2H), 3.92 (dd, lH, J 10 and 2.5 Hz), 3.86 (dd, lH, J 11 and 2 Hz), 3.77-3.56 (3H), 3.42 (dd, lH, J 9.5 and 8.5 Hz) 3.36 (s, 3H, CH30), 1.97 (s, 3H, CH3CO), 1.07 (d, 3H, J 6.5 Hz, Fuc-CH3).

(v) Methyl 2-acetamido-2-deoxy-6-0-~-L-fu~oy~LanoSyl-~-D-glucopyranoside (221 A solution of (21) (362 mg, 0.56 mmol) in acetic acid (50 ml) was hydrogenolysed at 230 kPa over 10% Pd/C (160 mg) over night. The mixture was filtered through a layer of Celite and concentrated. Column chromatography (chloroform-methanol-water, 65:40:10) of the residue gave amorphous (22) (192 mg, 90%,), t~]D22 -106 (c 1.1, H20).

lH NMR data (D20, CH30H ref., ~) : 4.95 ( d, lH, J 4 Hz, H-l'), 4.46 (d, lH, J 8.5 Hz, H-l), 4.15 (q, lH, J 6.5 Hz), 4.02 (dd, lH, J 12 and 1.5 Hz), 3.92 (dd, lH, J 10.5 and 3.5 HZ), 3.84-3.67 (4H), 3.62-3.49 (6H), 3.51 (s, CH30), 1.24 (d, 3H, J
6.5 Hz, Fuc-CH3).
13C NMR data (D20, CH30H ref., ~) : 177.7, lOS.0, 102.4, 78.0, 76.9, 74.8, 72.9, 72.5, 71.2, 70.3, 69.7, 60.0, 58.5, 25.2, 18.3.

SUB~ 11~ I.JTE SHEET

Wos5l~527 21 6 ~ 9 61 PCT/SE94/00604 3,3-Dl~thylbutyl 2-~cet~mi~o-2-deoYy-6-0-~-L-fucopyr~nosyl-~-D-glucolJ~.~noside ~24) r 5 (i) 3,3--Dimethylbutyl 3,4-di-0-acetyl-6-0-(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-2-deoxy-2-phthalimido-~-D-glucopyranoside (23) To a stirred mixture of (19) (8S3 mg, 1 mmol), 3,3 dimethylbutan-1-ol (0.182 ml, 1.5 mmol), N-iodosuccinimide (344 mg, 1.52 mmol) and powdered molecular sieves (0.9 g, 4 A) in dichloromethane-diethyl ether (2:1; 25 ml) at -30C, was added trifluoromethanesulphonic acid (0.017 ml, 0.19 mmol). After 1 h additional 3,3-dimethylbutan-1-ol (O.loO ml, 0.82 mmol) and trifluoromethanesulphonic acid (0.015 ml, 0.17 mmol) were added and stirring was continued for 2.S h. The reaction mixture was then filtered through Celite into an aqueous solution of sodium hydrogen carbonate and sodium bisulphite. The organic phase was w~cheA with agueous sodium chloride and concentrated. The residue was submitted to chromatography (ethyl acetate-heptane;
2:5) to give (23) (740 mg, 83%), t~]D22 -8.5 (c 1.3, CHCl3).

lH NMR data (CHC13, ~): 7.89-7.82 (2H), 7.78-7.70 (2H), 7.44-7.24 (15 H), 5.79 (dd, lH, J 11 and 9 Hz, H-3), 5.32 (d, lH, J 8.5 Hz, H-l), 5.08 (dd, lH, J 10 and 9 Hz, H-4), 4.99 and 4.66 (AB-system, 2H, J ll.S HZ, benzylic H), 4.91 (d, lH, J 3.5 Hz, H-l'), 4.88 and 4.77 (AB-system, 2H, J 12 Hz, benzylic H), 4.80 and 4.69 (AB system, 2H, J=12 Hz, benzylic H), 4.29 (dd, lH, J 11 and 8.5 Hz, H-2~, 4.05 (dd, lH, J 10 and 3.5 Hz), 3.99-3.73 (6H), 3.70-3.63 (2H), 3.38 ~m, lH), 1.98, (s, 3H, CH3CO), 1.87 (s, 3H, CH3C0), 1.30 (m; 2H, OCH2CH2), 1. 13 (d, 3H, J 6.5 Hz, Fuc-CH3), 0.69 (s, 9H).

Calc. for C51H49NO13: C 69.3; H 5.59; N 1.59. Found: C 68.4; H
6.65; N 1.75.

SU13~ ~ JTE SHEET

wos5loo527 PCT/SE94/00604 21 6~9 ~1 -58-(ii) 3,3-Dimethylbu~yl 2-acetamido-2-deoxy-6-o-~-L-fucopyranosyl-~-D-glucopyranoside (24) S Compound (23) (680 mg, ?6 mmol) was dissolved in methanol (25 ml). Methanolic sodium methoxide (0.2 M, 1 ml) was added and the mixture was stirred for 3.5 h. Neutralization with acidic cation exchange resin (Bio-Rad AG~ 50W-X8), filtration and evaporatiOn gave a residue which was dissolved in methanol (20 ml). Hydrazine monohydrate (O.S ml, 10.3 mmol) was added and the mixture was heated under reflux for 3.5 h and then cooled to 10C. Water (10 ml), methanol (2 ml) and acetic acid anhydride (2.5 ml) were added and the mixture was stirred at room temperature for 2.5 h during which time additional portions of acetic acid anhydride (2.0 and 0.5 ml) were added.
The methanol was then evaporated, and the aqueous residue was partitioned between dichloromethane and water. The aqueous phase was extracted with dichloromethane and the organic phase was concentrated. Chromatography of the residue (ethyl acetate-methanoli 20:1) gave a product which was dissolved in acetic acid (S0 ml). 10% Pd/C (160 mg) was added and the mixture was hydrogenolyzed at 230 kPa for 4 h at room temperature. The mixture was filtered through a layer of Celite and concentrated. Column chromatography of the residue (chloroform-methanol-water, 150:40:3~65:40:10) of the residue gave amorphous (24) (275 mg, 80%,), t~]D22 -87.2 (c 0.95, H20) .

lH NMR data (D20, CH30H ref., ~) : 4.94 (d, lH, J 4 Hz, H-l'), 4.S4 (d, lH, J 8.5 Hz, H-l), 4.15 tq, lH, J 6.5 Hz), 4.03-3.88 (8H), 2.03 (s, 3H, CH3CON), 1.58-1.40 (2H, OCH2CH2), 1.24 (d, 3Hr J 6.5 Hz, FUC-CH3), 0.90 (s, 9H).

13C NMR data (D20, CH30B ref., ~) : 177.S, 104.0, 102.4, 77.9, 76.9, 74.9, 73.0, 72.6, 71.2, 71.0, 69.7, 58.6, 45.0, 31.9, 25.2, 18.4.

SUB~3 111 ~JTE SHEET

W095/~7 21 6~9 61 PCT/SE94/00604 Calc. for C20H37NO1o~ C 53.2; H 8.26; N 3.10. Found: C 51.2; H

8.25; N 3.2.

~XAMP~E 7 ,, 5 Fuc~1-2~al~1-O-spacer ~-~8A ~31) ..
i) Ethyl 2-0-acetyl-3,4,6-tri-0-benzyl-1-thio-~-D-galactopyranoside (25) Compound (25) was prepared from acetobromogalactose (70.73 g, 0.172 mmol), according to proceedure described by S Nilsson, H
L~nn and T Norberg, Glycoconjugate J., 1989, 6, 21-34. Yield of (25) wa$ 26.13 g t28%).
TLC: Rf 0.33 (heptane:ethyl acetate, 9:2) 13C-NMR (CDC13) ~: 170.2 (CO), 139.2, 138.6, 138.4 (aromatic C), 84.2, 82.1 78.1, 75.0, 74.1, 73.6, 72.6, 70.3, 69.2, (C-1,2,3,4,5,6, 3x CH2Ph), 24.1 (SCH2CH3), 21.6 (OCOCH3), 15.4 (SCH2CH3~.

lH-NMR (CDCl3) ~: 5.43 (bt, lH, J2,3 9-7 Hz, H-2), (d lH~ Jl,2 11.9 Hz, H-l), 4.01 (bd, lH, J3,4 2.9 Hz, H-4), 3.55 (dd, lH, H-3).

(ii) Ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-l-thio-~-D-galactopyranoside (26) Ethyl 2-O-acetyl-3,4,6-tri-O-~enzyl-l-thio-~-D-galactopyranoside (25) was deacetylated with sodium methoxide ~n methanol (50 ml, pH 12) and subsequently benzoylated with benzoylchloride (1.96 gr., 14 mmol) in pyridine (20 ml) according to st~Ard procedures. Crystalline 26 was obtained in almos~ quantitative yield (3.44 gr., 97%).

NMR (CDCl3) lH: ~ 5.70 (lH, t, 9.8Hz, H-2) 2.60-2.80 (2H, m, -CH2cH3) ~

SIJB~ ~ JTE SHEET

WO 95t~S27 2 1 6 4 9 6 I PCT/SE94/00604~

13C: ~ 14.8, 23.6 (~Et), 68.6, 70.2, 71.7, 72.8, 73.6, 74.4, 76.6, 127.5-138.6 (aromatic C), 165.4 (C=O) (iii) 2-~zidoethyl 3,4,6-tri-O-benzyl-~-D-galactOPYranoside (28) To a stirred suspension of the thioglycoside (26) (700 mg, 1.17 mmol), 2-azidoethanol (204 mg, 2.34 mmol; prepared according to A. Ya. Chernyak et al. and A.V. Rama Rao, Carbohydr. Res., 1992, 223, 303-309), N-iodosuccinimide (395 mg, 1.75 mmol,) and ground molecular sieves (3A, 400 mg) in dichloromethane (25 ml) was added at 0C trifluoromethanesulfonic acid (TfOH; 35 mg, 0.23 mmol; according to method published by G.H. Veeneman, S.H.
Van Leeuwen, J.H. Van Boom, Tetrahedron Lett., 1990, 31, 1331).
When TLC (toluene:ethyl acetate, 6:1) showed complete conversion ( ~ 15 minutes), reaction was quenched by addition of triethylamine at 0C. The solution was filtered through a layer of celite, diluted with dichloromethane and washed twice with aqueous Na2S2O3 (10~) and finally with water.
The organic phase was dried over magnesium sulfate, filtered and concentrated and the residue was immediately subjected to TLC (toluene:ethyl acetate, 15:1). Solvent removal left 672 mg of 2-Azidoethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-~-D-galactopyranoside (27) as a colourless oil (92%), which was treated with sodium methoxide in methanol (pH 11) at room temperature for 6 hours. The solution was neutralized with Dowex 50 H+ resin, filtered and concentrated. The crystalline product (28) (540 mg, 89% from 2) was used without further purification for the preparation of disaccaride (29).
Compound (27): NMR (CDC13) lH: ~5.66 (lH, dd, 10.0, 7.9 HZ, H-2) 4.57 (lH, d, 7.8 Hz, H-1) 13C: ~ 50.7 (CH2N3, 67.3, 68.7, 71.9, 72.5, 73.6, 73.9, 74.5, 101.4 (C-l), 127.6-137.8 (aromatic C), 165.3 (C=O) Compound (28): 13C: ~ 50.7 (CH2N3), 68.4, 68.7, 71.4, 72.6, 73.0, 73.6, 73.9, 74.5, 81.7, 103.4 (C-1), 125.3-138.4 (aromatic C) SUB~ ITE SHEEi-=
wo9s/~27 216 4 9 61 pcTlsæ94loo6o4 (iv) 2-Azidoethyl 3,4,6-tri-0-benzyl-2-0-(2,3,4-tri-0-benzyl-~-L-fucopyranosyl)-~-D-galactopyranoside (29) To a solution of thioethylglycoside (14) (400 mg, 0.836 mmol) - 5 in dichloromethane (10 ml), bromine (134 mg, 0.836 mmol) was added at 0C. After about S minutes at 0C, the solution was allowed to attain room temperature and the solvent was evaporated. After co-evaporation with toluene the residue was dissolved in dichloromethane (2 ml) and added at room temperature to a suspension of tetraethylammonium bromide (176 mg, 0.835 mmol; prepared according to R.U. Lemieux, K.B.
Hendriks, R.V. Stick, K. James, J. Am. Chem. Soc. 1975, 97:14, 4056), compound (28) (290 mg, 0.558 mmol) and ground molecular sieves (3A, 300 mg) in CH2Cl2:DMF (4:1, 7 ml). TLC
(toluene:ethyl acetate, 6:1) showed complete conversion after stirring for 20 hours. The mixture was filtered, diluted with dichloromethane and washed with water. The organic phase was dried over magnesium sulfate and filtered and concentrated in vacuo. Preparative TLC yielded the title compound (29) as a viscous oil (407 mg, 78~).

NMR (CDC13) 13C: ~ 16.5, 33.6, 50.9, 66.4, 66.9, 68.8, 71.4, 72.0, 7208, 72.9, 73.5, 73.6, 74.4, 74.8, 75.7, 78.1, 79.6, 84.3, 97.3, 102.0, 125.3-129.0 (aromatic C), 138.0, 138.3, 139Ø

(v) 2-Aminoethyl 2-o-~-L-fucopyranosyl-~-D-galactopyranoside (30) The protected disaccaride derivative (29) (80 mg, 85 ~mol) was dissolve~ in ethanol (abs., 10 ml) and water (1 ml) and Pd/C
(10%, lO0 mg) was added. The mixture was hydrogenated and stirred rapidly at room temperature at 50 PSI. When reaction was not completed within 60 hours, the mixture was filtered and the product formed was isolated (TLC, ethyl acetate:~ethanol:acetic acid:water, 5:3:3:1, Rf=O.lS). After concentration in vacuo, the residue was resolved in a buffer of aqueous pyridine/acetic acid (2.5%/1%, pH 5.4) and eluated r SUBS 111 ~JTE SHEET

through a Bio Gel P-2 column. Evaporation and freeze drying gave 14 mg (44%) of the title compound (30) as a white powder.

NMR (CDC13) 13C: ~ 16.8, 39.8, 61.1, 66.4, 67.1, 68.7, 69.6, 71.9, 73.0, 7s.0, 78.4, 100.0, 101.7 (vi) Fuc~1-2Gal~l-O-spacer 4-HSA (31) To a stirred ice-cooled solution of thiophosgene (10 eq.) in tetrahydrofuran t2 ml), the amino derivative (30) (30 ~mol) in sodium borate buffer (0.85 M, 2 ml, pH 8.5) was added. The solution was stirred at room temperature for 10 minutes and then extracted with diethylether (3 x 2 ml). The aqueous phase containing the isothiocyanate derivative was added to a solution of Human Serum Albumine (HSA) (1/30 eq.) in the same buffer system (0.5 ml). pH was adjusted to 8.5 with aqueous sodium hydroxide (0.25 M) and the mixture was stirred at room temperature for 48 hours. Freeze drying of the reaction mixture was followed by ultracentrifugation purification with Centriprep tubes (lOKO). Freeze drying of the purified solutions gave the ~SA-conjugates (31) in excellent yield (18 mg).The degree of substitution was determined by Time of Flight masspectroscopy to 8 mol disaccharide/mol protein.

EXAMPL~ 8 Fuc~1-2Gal~l-o-spacer -~8A ~36) (i) 8-Azido-3,6-dioxaoctyl 3,4,6-tri-O-benzyl-~-D-galactopyranoside (33) The azidoderivative (33) was synthesized from the thioglycoside t26) (1004 mg, 1.68 mmol) and 1-azido-8-hydroxy-3,6-dioxaoctane (686 mg, 3.35 mmol; prepared according to C.R. Bertozzi, M.D.
R~nArski, J. Org. Chem., 1991, 56, 4326-4329) according to a procedure similar to the one used for synthesis o~ derivative ~28) (TLC; toluene:EtOAc 6:1) showed complete conversion within 40 minutes. Similar workup and deacylation of 8-azido-3,6-SlJBs I ~ I JTE SHEEI

W095/00527 2 1 6 ~ 9 6 ~ PCT/SE94/00604 dioxaoctyl 2-o-benzoyl-3,4,6-tri-0-benzyl-~-D-galactopyranoside (32) yielded 933 mg (78~ from 26) of the title compound (33) as a viscous ~

-~- 5 Compound (32); NMR (CDC13): 1H: ~ 5.64 (lH, dd, 10.0, 7.9 Hz, H-2).
13C: ~ 50.6 (CH2N3), 68.7, 68.9, 69.8, ~0.3, 70.5, 70.7, 71.8, 71.9, 72.6, 73.6, 73.8, 74.6, 80.0, 101.6 (C-l) Compound (33); 13C:~ 50.6 (CH2N3), 68.7, 68.8, 70.0, 70.2, 70.5, 7~.6, 71.4, 72.6, 73.3, 73.5, 73.8, 74.5, 81.9, 103.8 (C-1) (ii) 8-Azido-3,6-dioxaoctyl 3,4,6-tri-0-benzyl-2-0-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-~-D-galactopyranoside (34) Disaccaride (34) was synthesized from compound (33) (500 mg, 0.82 mmol) and thioethylglycoside (14) (512 mg, 1.07 mmol) according to the proce~llre described for the corresponding derivative (29). Preparative ~LC gave 683 mg (81%) of the title compound (34) as an oil.

MMR (CDCl3) 13C: ~ 18.3, 50.2, 66.2, 68.2, 68.8, 70.0, 70.2, 70.3, 70.6, 71.2, 72.0, 72.3, 72.5, 73.0, 73.3, 73.6, 74.4, 74,6, 75.8, 78.0, 79.7, 84.2, 98.6, 102.0 (iii) 8-~mino-3,6-dioxaoctyl 2-0-~-L-fucopyranosyl-~-D-galactopyranoside (35) 8-Azido-3,6-dioxaoctyl 3,4,6-tri-0-benzyl-2-0-(2,3,4 tri-O-benzyl-~L-fucopyranosyl)-~-D-galactopyranOSide (34) (35 mg, 34 ~mol) was dissolved in a mixture of ethyl acetate:ethanol:water in 1:2:2 (vol., 12 ml) and acidified with 20 ~l HOAc (according to method published by S. Nilsson, Doctoral dissertation, Lund University, April 1992). The solution was hydrogenated at 50 PSI on 10% Pd/C (140 mg) at room temperature overnight and when TLC (ethyl acetate:methanol:acetic acid:water, 5:3:3:1) showed complete deprotection, the mixture SUB~ JTE SHEET

wos~/~s27 PCT/SE94/00604 ~16~G~-64- --was filtered and evaporated. Purification on a Bio-Gel~ P-2 column taq. pyridine:acetic acid, 2.5:1 by vol., pH 5.4) concentration and freeze drying gave the title compound (35) as a white powder (14 mg, 90%).
NMR (CDC13) 13C: ~ 15.2 (CH3), 38.9, 60.7, 66.1, 66.5, 68.1, 68.s, 68.7, 69.2, 69.3, 69.4, 69.6, 71.7, 73.4, 74.8, 76.6, 99.2 (C-l'), 101.4 (C-1).

tiV) Fuc~1-2Gal~1-o-spacer 1-HSA (36) To a stirred ice-cooled solution of thiophosgene (10 eq.) in tetrahydrofuran (2 ml), the amino derivative (35) (30 ~mol) in sodium borate buffer (0.85 M, 2 ml, pH 8.5) was added. The solution was stirred at room temperature for lO minutes and then extracted with diethylether (3 x 2 ml). The aqueous phase containing the isothiocyanate derivative was added to a solution of Human Serum Albumine (HSA) (1/30 eq.) in the same buffer system (0.5 ml). pH was adjusted to 8.5 with aqueous sodium hydroxide (0.25 M) and the mixture was stirred at room temperature for 48 hours. Freeze drying of the reaction mixture was followed by ultracentrifugation purification with Centriprep tubes (lOKO). Freeze drying of the purified solutions gave the HSA-conjugates 36 in excellent yields (33 mg).
The degree of substitution was determined by Time of Flight masspectroScopy to 5 mol disaccharide/mol protein.

EXA~P~E 9 Fuc~-2Gal~1-o-~pacer 2-~AA (38) (i) 8-N-acrylamido-3.6-dioxaoctyl 2-0-~-L-fucopyranosyl-~-D-galactopyranoside (37) 0.8 ml deaerated 0.5 M sodiumborate aq. buffer (pH 8.5) and 2.4 ml deaerated methanol was added to 15 mg of the compound (35).
The reaction mixture was flushed with nitrogen and cooled to SUB~ JTE SHEET

W095l~5~ 2 1 6 ~ 9 6 ~ PCT/SE94/00604 0C. 3.3 ~1 of acryioylchloride was added and stirring was continued for 10 minutes. The reaction mixture was concentrated at room temperature to about a third of its original volume.
Purification on a Bio-Gel- P2 column and lyophilization gave S the title compound (37) ~14 mg, 83%) , NMR-data: 13C (D2O): ~ 15.0 (CH3), 38.54 (CH2N), 60.51, 66.31, 67.87, 68.19, 68.30, 68.50, 68.98, 69.10, 69.12, 69.43, 71.50, 73.25, 74.55, 76.08 (C-2,3,4,5,6; C-2,3,4,5; 5xCH2O) 98.88 (C-1'), 101.16 (C-l), 126.92 and 129.43 (CH=CH2).

(ii) Fuc~1-2Gal~l-O-spacer 2-PAA (38) To a solution of the compound (373 (14 mg, 0.027 mmol) and acrylamide (9.7 mg, 0,14 mmol) in deaerated water (1 ml) was added first N,N,N~N~-tetramethylendiamine (6 ~1) and then ammonium persulphate (3.5 mg). The mixture was stirred at roomtemperature over night. The polymer (38) obtained was purified by gel chromatography on a Bio-Gel- P2 column.
Freeze-drying of the purified solutions gave the PAA conjugate in excellent yield (17.9 mg). lH-NMR showed an average i..~oL~oration of 1 oligosaccharide per 7 acrylamide units.
.

E~MPr~E 1 0 FUC~1--2G~ 3 ~FUCt~ )GlCNAC,B--3G~l,~ Glc,Bl--N~l--PAA (~2) (i) Fuc~l-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-4Glc~l-NH2 (40).
Solid ammonium bicarbonate was added until æaturation to a solution of Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-4Glcl-OH
(Lewis B hexasackaride (39), purchased from Iso Sep AB, 25 mg in water (1.25 mL). The mixture was stirred in an open vessel at room kemperature for 6 days. Ammonium bicarbonate was added at intervals, saturation was assured by always keeping a portion of solid salt present in the mixture. When TLC
- 35 indicated no more conversion, the mixture was diluted with water (5 mL) and concentrated to half the original volume. The residue was diluted to 20 mL with water and concentrated to 5 mL. This process was repeated once, then the residue was SUBs ~ JTE SHEEr wos5/oos27 PCT/SE94/00604 21649~1 ~

diluted to 10 mL and lyophilized. The crude product was put on a Bio-gel P2-column, and the fraction containing Lewis B
glycosylamine (40) was collected, (20 mg 80~).

NMR data: 13C (D2O): ~ 84.66 (C-NR2), 97.54, 99.33, 100.39, 100.72, 102.98 (C-1 carbons of the nonreducing sugarunits), 15.08, 15.13 (2xCH3-fucose), 21.95 (CH3-CON-Glc~Ac).

(ii) Fuc~1-2Gal~1-3(Fuc~l-4)GlcNAc~1-3Gal~1-4GlC~1-NH-CO-CH-CH2 (41) Sodium carbonate (50 mg) and deaerated methanol (O.5 mL) was ~e~ to a solution of the glycosylamine (40) (20 mg, 0.02 mmol) in water (0.5 mL). The mixture was stirred at 0C while acryloyl chloride (60 ~L., 0.74 mmol) in tetrahydrofuran (0.5 mL) was added during 5 min. After 10 min. the solution was diluted with water (3 mL) and concentrated to 2 mL. The solution was again diluted with water (2 mL), 200 ~L
tetrahydrofuran (inhibitor solution) was added, and the solution was concentrated to 1-2 mL. This solution was purified by gel filtration on a Bio-Gel~ P2 column. Appropriate fractions were pooled and lyophilized to obtain the title compound (41) (14 mg, 67%).

NMR data: 13C (D2O): ~ 81.28 (C-NHCOCHCH2), 97.42, 99.18, 100.25, 102.59, 102.85 (C-1 carbons of the nonreducing sugaruni~s)~ 14.99, 15.06 (2xCH3-fucose), 21.92 (CH3CON-GlcNAc), 125.93, 130.32,(CH=CH2).
Fab ms: pseudomolecular ion m/z; 1053 (M+H) and 1075 (M+Na)+.
(iii) Fuc~1-2Gal~l-3(Fuc~1-4)GlcNAc~-3Gal~1-4GlC~l-NH-PAA (42) Copolymerization of N-Acryloylglycosylamine with acrylamide.
A solution of the N-acryloylglycosylamine (41) (13 ~mol) and acrylamide (53 ~mol, 3.7 mg) in distilled water (200 ~L) was deaerated by flushing with nitrogen for 20 min. The solution was then stirred at 0C and N,N,N,',N'-tetramethylethylenediamine (2 ~L) and ammonium persulfate (1 SUt~ ITE SHEET

W095/00527 21 ~ ~ 9 61 . ~ PCT/SE94100604 mg) wer~ added. The mixture was slowly stirred at 0C for 2 hours and then at room temperature overnight. The viscous solution was diluted with water (1 mL) and purified by gel filtration on Bio-Gel P2 column eluated with aqueous n-- 5 buthanol (1~). Fractions containing polymer were pooled and lyophilized. Yield: 3mg.

H-NMR ~ ws presence of approximately 1 Lewis ~ unit per 5 CHCH2 units.
ESANPL~ 11 Fuc1-2~ 3çT.~t~fll-o-~p~c~r 5-PAA (S0) (i) 2-azidoethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-~-D-glucopyranoside (43) Ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-~-D-glucopyranoside (prepared according to H L~nn, Carbohydr. Res., 139 (198S), 105-113) (O.S g, 1.1 mmol) was dissolved in 20 ml of dichloromethane and 2-azidoethanol (prepared according to Chernyak A.Y. et al. Carbohydr. Res., 1992, 223, (303-309) (0.148 g, 1.7 mmol) crushed 4A molecular sieves were added and the mixture stirred for 30 min. Dimethyl(methylthio)sulfonium 2S triflate (DMTST) (0.439 g, 1.7 mmol; prepared according to P.
F~gedi and P.J. Garegg, Carbohydr. Res., 149 (1989), 9-12) was added at room temperature and stirring was continued for 4 hours. Analysis by TLC (toluene-ethylacetate) show no starting material, and to the reaction mixture was added 1 ml of triethylamine and stirring was continued for another 30 min.
The reaction mixture was transfered to a silica gel column and eluted with toluene:ethylacetate 6:1 to give (372 mg, 72%) of the title compound (43).

NMR-data: 13C (CDC13): ~ 50.38 (CH2-N); 56.42 (CH-N); 66.2, (CH-O); 68.44 (CH2O); 68.50 (CH-O; 68.53 (CH2-O); 82.05 (CH-O);
98,89 (C-1); 101.83 (PhCH).

SUB~ JTE SHEET

wogs/00~7 PCT/SE94/00604 2l6~961_68- --(ii) 2-Azidoethyl 3-0-(2-0-acetyl-3,4,6-tri-0-benzyl-~-D-galcatopyranosyl)-4,6-o-benzylidene-2-deoxy-2-phthalimido-~-D-glucopyranoside (44) S Ethyl 2-0-acetyl-3,4,6-tri-0-benzyl-1-thio-~-D-galactopyranoside (25) (818 mg, 1.5 mmol) and the compound (43) (395 mg, 0.85 mmol) were dissolved in 30 ml of dichloromethane, crushed 4A molecular sieves were added and the mixture stirred for 20 min. The reaction was flushed with nitrogen and DMTST
(787 mg, 3.05 mmol, dissolved in 5 ml of dichloromethane) was added dropwise to the reaction mixture and the dropfunnel rinsed with 6 ml of dichloromethane. After 2 hours 1 ml of triethylamine was added and stirred for 30 min., filtration, concentration and column chromatography (toluene:ethylacetate 10:1 gave three fractions. Fraction 1 the ~-product (97.32, 98.89; (C-l and C-l'). Fraction 2 almost pure 44 (306 mg, 39~).

NMR-data: 13C (CDC13 ref. tetramethylsilane 0 ppm): ~ 20.32 (CH3C0), 50.47 (CH2N), S5.13 (CH-N), 66.57, 68.15, 68.20, 68.65, 71.58, 71.71, 72.17, 72.94, 73.46, 74.38, 75.15, 80.47, 81.08 (C-3,4,5,6 C-2,3,4,5,6; 3xCH2Ph; CH2-0), 98.86 (C-l), 100.75, 101.23 (C-l and CH Ph), 168.84 (C=0).

~iii) 2-azidoethyl 2-acetamido-3-0-(3,4,6-tri-0-benzyl-~-D-galactopyranosyl)-4~6-o-benzylidene-2-deoxy-~-D-glucopyranoside (45) To compound (44) (525 mg, 0.56 mmol) was added 50 ml of ethanol and 1.1 ml of hydrazinhydrate reflux over night and TLC
(toluene:ethylacetate 1:2) showed a new product. Concentration and coevaporation with toluene, followed and then dissolving in 45 ml of dichloromethane and washing with an equal amount of water, coevaporation with toluene, gave the crude monohydroxy amine. This crude product was dissolved in dichloromethane:methanol (1:1, 15 ml) and 1.5 ml of acetic anhydride was added. After 3 hours no starting material was left (TLC). Concentration and chromatography (toluene-SUB~ 111 ~JTE SHEET

W095/~527 ~ 6 1 PCT/SE94/00604 ethylacetate 1:2) ga~e (204 mg, 45~) of the title compound (45).

NMR-data: 13C (CDC13): ~ 23.59 (NHCOCH3), 50.59 (C-N), 56.89 (C-N), 6G.40, 68.28, 68.56, 70.55, 72.44, 73.21, 73.40, 73.53, 74.60, 76.08, 79.75, 81.71 (C-3,4,5,6; C-2',3',4',5',6';
3xCH2Ph; CH2O) 100.97, 101.25, 103.48 (C-1, C-11 and CHPh), 171.67 (C=O).

(iv) 2-azidoethyl 2-acetamido-3-O-(3,4,6-tri-O-benzyl-2-O-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-~-D-galctopyranosyl]-4,6-O-benzylidene-2-deoxy-~-D-glucopyranoside (46) The compound (45) (137 mg, 0.17 mmol) and the compound (14) (162 mg, 0.34 mmol) were dissolved in dichloromethane (75 ml), and molecular sieves (4A) were added, and the mixture was stirred for 20 min. DMTST (96 mg, 0.37 mmol) was added and stirring was continued for 1.5 hours. 1 ml of triethylamine was added and stirring was continued for another 20 min. Filtration through celite, concentration and column chromatography (toluene:ethylacetate 1:1) gave (46) (101 mg, 49%).

NMR-data: 13C (CDC13): ~ 16.83 (C83 fucose), 23.30 (NHCOCH3), 50.66 (CH2-N), 57.40 (C-2), 66.55, 67.17, 67.96, 68.60, 72.31, 72.91, 72.99, 73.04, 73,10, 73.51, 74.48, 74.6s, 76.05, 76.29, 76.62, 77.60, 79.43, 79.53, 83.21 (C-3,4,5,6; C-2',3',4',5~,6~;
C-2", 3",4",5"; 6xCH2Ph), 97.67 (C-l"), 100.94, 101.07, 102.13 tC-1, C-l', CHPh), 170.92 (C=O).

(v) 2-trifluoracetamidoethyl 2-acetamido-3-O-t3,4,6-tri-O-benzyl-2-o-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-~-D-galactopyranosyl]-4~6-o-benzylidene-2-deoxy-~-D-glucopyranoside (47).

The compound (46) (135 mg, 0.11 mmol) was dissolved in 11 mL of ethanol and 10% Pd/C (140 mg) was added. The reaction mixture was hydrogenated at atmospheric pressure for 15 minutes.
Analysis by Tlc (ethyl acetate:methanol:acetic acid:water SUB~ JTE SHEET

W095/00~7 PCT/SE94/00604 216~9~1 ~70- -12:3:3:1) showed no starting material, but one ninhydrin positive product. The mixture was filtered through celite, concentrated and dissolved in dichloromethane (7 mL), and pyridine (3.5 mL), flushed with nitrogen and cooled to 0C.
Trifluoroacetic anhydride (31 ~1, 0.22 mmol) was added. After one hour the mixture was concentrated and coevaporated with 2 ml of toluene twice. Column chromatography (toluene:ethyl acetate, 1:3) gave 47 (73 mg, 52%) NMR-data: 13C (CDC13): ~ 17.06 (CH3 fucose) 22.66 (NHCOCH3), 39.59 (CH2-N), 54.83 (C-2), 65.56, 66.77, 67.78, 68.44, 68.69, 72.80, 73.05, 73.24, 2x73.46, 74.46, 74.66, 76.38, 77.08, 77.80, 78.91, 79.96, 80.01, 82.07, (C-3,4,5,6; C-2',3',4~,S~,6~; C-2",3",4",5"; 6xCH2Ph) 98.61 (C-1"), 101.22, lol.9s~ 102.35 (C-l, C-l~, CHPh), 171.64 (NHCOCH3).

(vi) 2-trifluoroacetamidoethyl 2-acetamido-2-deoxy-3-O-t2-O-(~-L-fucopyranosyl)-~-D-galactopyranosyl]-~-D-glucopyranoside (48) Trisaccharide (47) (73mg, S6.2 ~mol) was dissolved in absolute ethanol (7 ml) with water (0.25 ml) and glacial acetic acid (2 ~1). The solution was hydrogenated over 10% Pd/C (152 mg) at 50 PSI at room temperature for 1 hour. When TLC (ethyl acetate:acetic acid:methanol:water 12:3:3:1; R~ = 0.14 for the compound (48) showed complete conversion, the reaction mixture was filtered through a layer of celite and concentrated. The crude, solid residue (46 mg) was used in the next reaction without further purification.

NMR-data: 13C (D2O): ~ 16.59 (CH3 fucose), 21,85 (NHCOCH3), 39.44 (CH2-N), 54.56 (C-2), 60.45-76.9S (C3,4,5,6, C2',3',4',s',6', 2",3",4",5") 99.29, 99.92, 101.28 (C-1, C-1', C-l''), 173.48 (NHCOCH3).

SUB~ 111 ~TE SHEET

woss/~527 ~ 61 PCT/SE94/00604 -71- ~
(vii) 2-acrylamidoethyl 2-acetamido-2-deoxy-3-0-2-0-~-L-fucopyranosyl-~-D-galactopyranosyl-~-D-glucopyranoside (49) The crude compound (48) (46 mg) was dissolved in aqueous ~-~ 5 ammonia (25%, 4 ml) and stirred at room temperatur. The reaction was complete within 1 hour and yielded the free amino ~ derivat~ve exclusively. (TLC ethy~ acetate:acetic acid:methanol :water 5:3:3:1). Cor.centration and co-concentration with toluene was followed by purification on a Bond-Elut (SCX, H~-form) cation exchange resin 0.5 g cartridge. The sample was dissolved in 3 ml of water and pH was adjusted to pH 6 with aqueous acetic acid. The sample was put on the column and then eluted with 2M ammonia in methanol:water, 1:1 (5 ml). The fractions containing free amine (ninhydrin positive) were pooled, concentrated and lyophilized to give (30 mg, 0.05 mmol) crude amine.

1 ml deaerated 0.5 M sodiumborate (aq buffer (pH 8.5) and deaerated methanol (3 ml) was added to the crude amine. The reactio~ mixture was flushed with nitrogen and cooled to 0C, 6.4 ~1 (0.078 mmol) acryloylchloride was added and stirring was continued for 10 minutes. The reaction mixture was concentrated at room temperature to about a third of its original volume.
Purification on a Bio-Gel P2 column and lyophilization gave 49 of the title compound (49) (30 mg, 86~ from (47)).

NMR-data: 13C (D20): ~ 14.99 (-CH3, fucose), 21.99 (NHCOCH3), 39.10 (CH2N), 54.58 (C-2) 99.25, 99.93, 101.39 (C-1, C'-1, C"-1), 127.27, 129.65 (CH=CH2).
(viii) Fuc~1-2Gal~1-3GlcNAc~1-0-spacer 5-PAA (50) Copolymerization of 2-acrylamidoethyl 2-acetamido-2-deoxy-3-o-2-o-~-L-fucopyranosyl-~-D-galactopyranosyl-~-D-glucopyranoside (49) with acrylamide.

To acrylamide (10 mg, 144 ~mol) was added at room temperature a solution of the trisaccharide (49) (18 mg, 29 ~mol) in SUB~ JTE SHEEr Wo 9~/00s27 PCT/SE94/00604 2l~4~ 72- --deaerated water (1 ml). To this slowly stirred solution (kept in the dark and under nitrogen) was added at oC~ first N,N,N~,N~-tetramethylethylenediamine (6 ~1), and then ammonium persulphate (3.5 mg). The mixture was stirred at room 5 temperature over night. TLC (ethyl acetate:acetic acid:methanol :water 5:3:3:2) showed that almost all of the compound (49) was consumed and that a charring baseline product had been formed.
The polymer was purified by gel chromatography on a Bio-Gel P-2 column eluted with aqueous n-butanol (1%). Freeze-drying of 10 the polymeric fraction eluted in the void volume gave 13.1 mg of the polymer (So) were the lH N~ analysis of the product showed an average incorporation of 1 trisaccharide per 7.6 acrylamide units, and 11.9 mg of polymer (50) were the lH NMR
analysis of the product showed an average incorporation of 1 15 trisaccharide per 10.3 acrylamide units.

B~ PI~IS 12 Fuc~l-2Gzll~Bl-3lFuc~ )alGNAc~Bl-o-sp~c~r 5--PAA(55) (i) 2-azidoethyl 2-acetamido-6-0-benzyl-3-0-t3,4,6-tri-0-benzyl-~B-D-galactopyranosyl)-2-deoxy-~B-D-glucopyranoside (51).

Diethyl ether saturated with hydrogen chloride was added, at 25 roomtemperature, to a stirred mixture of 2-azidoethyl 2-acetamido-3-o-3~4~6-tri-o-benzyl-~B-D-galactopyranosyl-4~6 benzylidene-2-deoxy-,~-D-glucopyranoside (45) (420 mg, 0.52 mmol), sodium cyanoborohydrids (200 mg, 3,2 mmol) and molecular sieves 3A in tetrahydrofuran (20 ml) until the mixture was 30 acidic (as determined with indicator paper; method according to M. Nilsson and T. Norberg Carbohydr. Res., 183 (1988 71-82).
The mixture was stirred for 20 min. at roomtemperature and then triethylamine (0.30 mL) was added. The mixture was filtered through Celite, washed with water, dried and evaporated. The 35 crude product was purified by column chromatography (toluene:
ethyl acetate, 6:1) to give pure compound (51) (266 mg, 0.32 mmol, 65%).

SIJB-~ 1 l l ~JTE SHEET

W095/~527 2 1 6 ~ 9 6 f PCT/SE94/00604 NMR-data: 13C (cDCl,): ~ 23.41 (NHCOCH3), 50, 30 (CH2N), 56, 81 (C-N), 66,3-81,9 (C-3,4,5,6; C-2',3',4',5',6'; 4xCH2Ph; CH2O) 100.90, 103.21 (C-1, C-l'), 173,4 (CO) (ii) 2 azidoethyl 2-acetamido-2-deoxy-4-0-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-3-O-t3,4,6-tri-O-benzyl-2-O-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-~-D-galactopyranosyl]-~-D
glucopyranoside (52).
The compound (51) (157 mg, 0.19 mmol) and the compound (14) (362 mg, 0.76 mmol) were dissolved in dichloromethane (100 ml), and 3g ~A molecular sieve (MS) were added and stirred for 20 min. Dimethyl (methylthio)sulfonium triflate (DMTST) (207 mg, 0.80 mmol) was added and stirring waæ continued for 1.5 hour. 2 ml of triethylamine was added and stirring was continued for another 20 min. Filtration through celite, concentration and column chromatography (toluen:ethylacetate, 1:1) gave the title compound (52) (142 mg, 0.086 mmol, 45%).
NMR-data: 13C (CDC13): ~ 17.01, 16.81 (2xCH3 fucose), 23.20 (NHCOCH3), 50.35 (CH2-N), 57.21 (C-2), 98.31, 99.70, 101.14, 102.30 (C1, C1', 2xC1-fucose), 170.30 (C=O).

(iii) 2-trifluoroacetamidoethyl 2-acetamido-2-deoxy-4-O-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-3-0-~3,4,6 tri-O-benzyl-2-O-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-~-D-galactopyranosyl]-~-D-glycopyranoside (53).
The compound (52) (140 mg, 0.084 mmol) was dissolved in 11 ml ethanol and 10% Pd/C (150 mg) was added. The reaction mixture was hydrogenated at atmospheric pressure for 15 minutes.
Analysis by TLC (ethyl acetate:methanol:acetic acid:water 12:3:3:1) showed no starting material, but one ninhydrin positive product. The mixtures was filtered through celite, concentrated and dissolved in dichloromethane (10 ml) and pyridine (3.5 ml), flushed with nitrogen and cooled to 0C.

SUB~ JT~ SHEET

wos5/00527 PCT/SE94/00604 2 1 6~ 6~ _74_ Trifluoroacetic anhydride (31 ~1, 0.22 mmol) was added. After one hour the mixture was concentrated and coevaporated with 2 ml of toluene twice. Column chromatography (toluene:ethyl acetate 1:2~ gave the compound (53) (82.8 mg, 0.053 mmol, 63%).
.
NMR-data: 13C (CDC13): ~ 17.33, 16.93 ( 2xCH3 fucose), 22.30 (NHCOCH3), 39.25 (CH2-N), 54.48 (C-2), 99.03, 99.98, 101.63, 102.75, (Cl, Cl', 2xCl-fucose), 171.73 (NHCOC~3).

(iv) 2-trifluoroacetamidoethyl 2-acetamido-2-deoxy-3-0-(2-O-~-L-fucopyranosyl-~-D-galactopyranosyl)-4-o-~-L-fucopyran D-glucopyranoside (54).

The tetrasaccharide (53) (78 mg, 0.05 mmol) was dissolved in absolute ethanol (8 ml) with water (0.25 ml) and glacial acetic acid (2 ~L). The solution was rapidly stirred with 10~ Pd/C
(150 mg) under hydrogen (50 PSI) at room temperature for 1 hour. When TLC (ethyl acetate:acetic acid:methanol:water 12:3 3:1; showed complete conversion, the reaction mixture was filtered thorugh a layer of celite and concentrated. The crude compound (54) (35 mg) was used in the next reaction without further purification.

NMR-data: 11C (CDC13): ~ 16.91, 16.53 (2xCH3 fucose), 22.15 (NHCOCH3), 39.14 (CH2N), 54.20 (C-2), 99.33, 100.03, 101.73, 102.95 (C-l, C-l', 2xC-1 fucose), 173.30 (NHCOCH3) there were no 13C signals in the "aromatic region".

(v) 2-acrylamidoethyl 2-acetamido-2-deoxy-3-O-(2-O-(~-L-fucopyranosyl-~-D-galactopyranosyl)-4-O-~-L-fucopyranosyl-~-D-glucopyranoside (5S).

35 mg of the crude compound (54) was dissolved in aqueous ammonia (25%, 4 ml) and stirred at room tPmrPrature. The reaction was complete within 1 hour and yielded the free amino derivative exclusively. TLC (ethylacetate:acetic acid:
methanol:water, 5:3:3:2), concentration and co-concentration SUB~ 111 JTE SHEET

wos5l0o~27 PCTISE94/00604 ~ 2l6~96I_75 ''~

with toluene was foilowed by purification on a Bond-Elut cartridge (SCX, H+-form) cation exchange resin. The sample was dissolved in 3 ml of water and pH was adjusted to 6 with aqueous acetic acid. The sample was put on the column and then eluted with 2M ammonia in methanol:water, 1:1 (5 ml). The fraction~ containing free amine (ninhydrin positive) were pooled, concentrated and lyophilized to give crude amine (20 mg). 1 ml Deaerated 0.5 M sodiumborate (aq) buffer (pH 8.5) and deaerated methanol (3 ml) was added to the crude amine. The reaction mixture was flushed with nitrogen and cooled to 0C. 6 ~L acryloylchloride was added and stirring was continued for 10 min. The reaction mixture was co~c~ntrated at room temperature to about a third of its original volume. Purification on a Bio-Gel- P2 column and lyophilization gave pure title compound (55) (15 mg).

NMR-data: 13C (D2O): ~ 16.90, 16.45 (2xCH3 fucose), 21.95 (NHCOCH3) 39.51 (CH2N), 54.31 (C-2) g9.21, 99.95, 101.56, 102.87 (C-l, C-l', 2xC-1 fucose), 127.21, 129.57 (CH=CH2) 173.27 (NHCOCH3).

(vi) Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~l-O-spacer 5-PAA (56) Copolymerization of 2-acrylamidoethyl 2-acetamido-2-deoxy-3-O-2-O-~-L-fucopyranosyl-~-D-galactopyranosyl-4-~-L-fUcopyranosyl-~-D-glucopyranoside (55) and acrylamide.

To acrylamide (8.3 mg, 120 ~mol) was added at room temperature a solution of tetrasaccharide (54) tl5 mg, 20 ~mol) in deaerated water (1 ml). To this slowly stirred solution (kept in the dark and under nitrogen atmosphere was added at 0OC, first N,N,N',N'-tetramethylethylenediamine (6 ~1), and then ammonium persulphate (3.5 mg). The mixture was stirred at room temperature over night. TLC (ethyl acetate:acetic acid:methanol :water 5:3:2) showed that all of compound (49) was consumed and that a charring baseline product had been formed. The polymer was purified by gel chromatography on a Bio-Gel P-2 column SUB~ JTE SHEEl-Woss/oo~27 2 1 6 ~ 9 G 1 PCTISE94/00604 eluted with aqueous n-butanol (1%). Freeze-drying of the polymeric fraction eluted in the void volume gave 20 mg of polymer (s6).

S A lH-NMR analysis of the product showed an average incorporation of 1 trisaccharide per 6 acrylamide units.
., ~XAMP~ 3 Fuc~1-2 Gal~1-O-~F~ S-PAA (58) (i) 2-acrylamidoethyl 2-O-~-L-fucopyranosyl-~-D-galactopyranoside (57) lS 0.3 ml deaerated 0.5 M sodiumborate (aq) buffer (pH 8.5) and methanol (0.9 ml) was added to 6.4 mg of the compound (30). The reaction mixture was flushed with nitrogen and cooled to 0C.
2 ~1 acryloyl chloride was added and stirring was continued for 10 minutes. The reaction mixture was concentrated at room temperature to about a third of its original volume.
Purification on a Bio-Gel~ P2 column and lyophilization gave the compound (57) (4 mg, 57%).

NMR-data: 1H (D2O): ~ 1.2 (d, CH3 fucose) 4.52 (dd, H-l), s.22 (m, H-l'), S.80 (dd, CH=CH2), 6.25 (m, CH=CH2).

(ii) Fuc~1-2 Gal~1-O-spacer 5-PAA (58) To a solution of the compound (57) (4 mg, 9 ~mol) and acrylamide (3.3 mg, 47 ~mol) in deaerated water (0.7S ml) was added first N,N,N',N'-tetramethylenediamine (2 ~1) and then ammonium persulphate (1.5 mg). The mixture was stirred at room temperature over night. The polymer (58) was purified on a Bio-Gel P2 column (9.1 mg).

NMR-data: lH (D20) showed an average incorporation of 1 oligosaccharide per 12.3 acrylamide units.

SUI~s 111 ~JTE SHEE~

WO 95/00527 21 6 ~ 9 61 PCT/SE94/00604 BIOLOGICAL EXPERIMENTS

Materials and Methods - S In situ adherence assay for Helicobacter pylori Non-infected samples from normal adult human gastric tissue (o~tained from Huddinge Sjukhus, Sweden) were used to study Helicobacter pylori adherence. All samples were fixed in 4%
formalin and subsequently e~h~ed in paraffin.

Sect~ons, 4 ~m thick, were placed on glass slides and used for Steiner~s silver staining (to identify the cell types present in gastric units, and to verify that the tissue samples have no pathologic changes) and/or subsequent adherence assay.

Four clinical isolates, A4, A5, A7, and A8 (obtained from H~ inge Sjukhus) of Helicobacter pylo~i were used.
Nelicobacter pylori was cultured at 37C on Brucella Agar supplemented with 10% bovine blood and l~ IsoVitalex (Becton Dickinson Microbiology System, Cockeyville, MD) under microaerophilic conditions (5~ 2~ 10% C02, 85% N2) and 98%
humidity. Five days after inoculation, bacteria from one full-grown plate were resuspended by gentle pipetting in 25 ml of 0.lM NaCl/ 0.lM sodium carbonate, pH 9Ø 250 ~l of a freshly prepared l0 mg/ml solution of fluorescein ~sothiocyanate (FITC, Sigma Chemical Co.) in dimethylsulfoxide was added to the suspension of bacteria which was then ~ h~ted for l hour at room temperature in the dark. The bacteria were recovered by centrifugation at 3000 x g for l0 minutes, and then resuspended in phosphate buffered saline (PBS) + 0.05% polyoxyethylene sorbitan monolaurate (Tween 20) by gentle pipetting and subsequently pelleted by centrifugation as aboveO The wash procedure was repeated 3 times and the ; 35 suspension was finally resuspended to an Optical Density of 0.2. The intensity of FITC-labelling of all bacterial strains was similar as ~udged by inspection of comparable numbers of organisms by fluorescence microscopy. Aliquots of l ml were SUB~ JT~ SHEET

W095/005Z7 pcTlsE9~l~aD~o1 2 ~ 78- ~
taken from the final suspensions and utilized immediately or stored at -20OC until use. No difference in binding pattern was observed between strains labelled and used immediately and strains that were frozen and thawed once before use.
Slide-mounted tissue sections were deparaffinized in Bio-Clear (Bio-Optica SpA) and absolute alcohol, 95% alcohol followed by 70S alcohol, rinsed in water followed by P8S and then incubated for 45 minutes in blocking buffer (1% gelatin/0.05% Tween 20 in PBS). FITC labelled bacterial suspension (OD about 0.200-0.2s0) was mixed with equal amount of a concentrated solution of the compound. The mixture was preincubated for 2 hours at room temperature in the dark, 200 ~l of the mixture was placed on a slide-mounted tissue section and incubated for 1 hour at room temperature in a humidified chamber. The slides were subsequently washed 6 times with PBS prior to inspection under fluorescence microscope.

y~li8 The in situ adherence assay was used to ascertain binding of Helicobacter pylori to human gastric tissue and to demonstrate inhibition of Helicobacter pylori with terminal L-fucose-cont~ining compounds, e.g. LNFl-HSA.

To analyze the ability of terminal L-fucose-containing compounds to inhibit binding. FITC labelled bacterial suspension tO.D. about 0.200-0.250) was mixed with equal amount of a concentrated solution of the compound. The mixture was preinc~lhAted for 2 hours at room temperature in the dark. 200~1 of the mixture was placed on a slide-mounted tissue section and was incubated for l hour at room temperature. After inc~hAtion, the treated tissue sections were washed 6 times with PBS before analysis of the tissue sections.

Comparison tissue sections treated with test compound with untreated tissue sections using fluorescence microscopy and image analysis (Neotech Image Grabber 24/1.1 to transfer the SUB~ l l l ~)TE SHEEr W095/~527 PCT/SE94/00604 6~9 61 -79-visual microscope ~mage to a computer screen and optilab 24/2.1.1 Grafted, to count the adhered bacteria).

The given values in the table are the average number of adhered ` 5 bacteria on three different areas per section comparing treated (with compound) with untreated tissue sections.

SU~ JTE SHEET

WO 95/005~7 PCT/SE!)~ G~50 1 ~6b~9~ -80- ~
T~ble (average value) tFuc~1-2Gal~1-spacer 1]s-HSA 2 mM 34%
"
tFucal-2Gal~l-spacer 4]8-HSA 2 mM 35%

tFuc~1-2Gal~1-spacer 2]n~PAA 2 mM 45%
n=1 per 12.3 acrylamide moieties tFUc~l-2Gal~l-spacer 5~-PAA 1 mM 53%
n=1 per 5 acrylamide moleties tFucal-2Gal~l-3GlcNAc~l-spacer 2]n-PAA 2 mM 40%
nsl per 7.6 acrylamide moieties [Fucal-2Gal~1-3GlcNAc~1-Gal~1-spacer 3]35--HSA (LNF1-HSA) 0.2 mM72%
tPurch~ from Iso Sep AB, Sweden) tFucal-2Gal~l-3(Fuc~l-4)GlcNAc~l-Ga~
-spacer 3]32-HSA (LND1-HSA) 0.2 mM71%
(Purchased from Iso Sep AB, Sweden) tFUcal-2Gal~1-3Fuc~1-4)GlcNac~l-Gal~1--spacer 3]n-PAA
n=1 per 18 acrylamide moieties 0.2 mM67~

n-1 per 5 acrylamide moieties 0.2 mM84%

n=1 per 6 acrylamide moieties 0.2 mM93%

tGalNAcal-3(Fucal-2)Gal~l-3(Fucal-4)- 0.2 mM80%
GlcNac~l-Gal~l-spacer 3]22-HSA (A-hepta-HSA) (Purchased from Iso Sep AB, Sweden) SUB~ JTE SHEEr

Claims (42)

1. Use of a compound of the general formula Ia, Ib, Ic, Id, Ie or If Ia Ib Ic Id Ie If wherein Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 independently are O, S, CH2, or NR25, where R25 is hydrogen, C1-24-alkyl, C2-24-alkenyl, C1-24-alkylcarbonyl, or benzoyl optionally substituted with hydroxy, amino, C1-4-alkyl, C1-4-alkoxy, nitro, halogen, phenoxy, or mono- or di-halogen-C1-4-alkyl;

Y is ; A is ;

B is ; C is ;
D is ; E is ;
wherein the wavy line in Y, A, B, C, D and E signifies a bond which is either in the .alpha.- or in the .beta.-configuration;

R1, R2, and R3 each independently are H, halogen, azido, guanidinyl, branched or unbranched C1-24-alkyl, C2-24-alkenyl, C2-24-alkynyl, C3-8-cycloalkyl, C3-8-cycloalkyl-C1-24-alkyl, or C1-12-alkoxy-C1-C12-alkyl group which is optionally substituted with hydroxy, amino, halogen, or oxo; aryl or aryl-C1-4-alkyl optionally substituted in the aryl moiety with hydroxy, amino, C1-4-alkyl, C1-4-alkoxy, nitro, halogen, phenoxy, or mono- or di-halogen-C1-4-alkyl;
tri(C1-4-alkyl)silylethyl; oxo;
a group =CR4R5 wherein R4 and R5 independently are H, or C1-4-alkyl;
or a group XR10 wherein X is O, S, NR20, or =N-, and R10 is H, branched or unbranched C1-24-alkyl, C2-24-alkenyl, C2-24-alkynyl, C3-8-cycloalkyl, C3-8-cycloalkyl-C1-24-alkyl, or C1-12-alkoxy-C2-12-alkyl group which is optionally substituted with hydroxy, amino, halogen, or oxo; aryl, aryl-C1-4-alkyl, or heterocyclyl-C1-4-alkyl optionally substituted in the aryl or heterocyclyl moiety with hydroxy, amino, C1-4-alkyl, C1-4-alkoxy, nitro, halogen, phenoxy, or mono-or di-halogen-C1-4-alkyl;
tri(C1-4 alkyl)silylethyl; tri(C1-4-alkyl)silyl;
tri(C1-4-alkyl)silylethoxymethyl; the acyl residue of a naturally occurring amino acid; C1-24-alkylcarbonyl;
C2-24-alkenylcarbonyl;
C3-8-cycloalkyl-C1-24-alkylcarbonyl; arylcarbonyl; or terpenyl; and R20 is H, C1-24-alkyl, C2-24-alkenyl, C1-24-alkylcarbonyl, or benzoyl or phthaloyl optionally substituted in the benzene ring with hydroxy, amino, C1-4-alkyl, C1-4-alkoxy, nitro, halogen, phenoxy, or mono- or di-halogen-C1-4-alkyl;

R1A, R2A, R3A, R4A, R1B, R2B, R3B, R4B, R1C, R2C, R3C, R4C, R1D, R2D, R3D, R4D, R1E, R2E, R3E, and R4E each independently is as defined for R1, R2, and R3 above, or is a group of the formula VII

wherein Y and Z1 are as defined above;
with the provisos that one of R1B, R2B, R3B, or R4B is Z3, Z5, Z8 or Z12, that one of R1C, R2C, R3C, or R4C is Z6, Z9 or Z13, that one of R1D, R2D, R3D, or R4D is Z10, or Z14, that one of R1E, R2E, R3E, or R4E is Z15, that at least one and at the most five of R1A, R2A, R3A, R4A, R1B, R2B, R3B, R48, R1C, R2C, R3C, R4C, R1D, R2D, R3D, R4D, R1E, R2E, R3E, and R4E is a group of the formula VII, and that the configurations of the substituents R1A, R2A, R3A, and R4ACH2 in A, the configurations of the substituents R1B, R2B, R3B, and R4BCH2 in B, the configurations of the substituents R1C, R2C, R3C, and R4CCH2 in C, the configurations of the substituents R1D, R2D, R3D, and R4DCH2 in D, and the configurations of the substituents R1E, R2E, R3E, and R4ECH2 in E independently are D-gluco, L-gluco, D-galacto, L-galacto, D-manno, L-manno, D-talo, L-talo, D-allo, L-allo, D-altro, L-altro, D-gulo, L-gulo, D-ido, or L-ido;

R is hydrogen, a branched or unbranched C1-24-alkyl, C2-24-alkenyl, C2-24-alkynyl, C3-8-cycloalkyl, C3-8-cycloalkyl-C1-24-alkyl, C1-12-alkoxy-C1-12-alkyl, C1-24-alkylcarbonyl, C2-24-alkenylcarbonyl, or C3-8-cycloalkyl-C1-24-alkylcarbonyl group which is optionally substituted with hydroxy, amino, halogen, or oxo; an aryl, aryl-C1-4-alkyl, arylcarbonyl or aryl-C1-4-alkylcarbonyl group optionally substituted in the aryl moiety with hydroxy, amino, C1-4-alkyl, C1-4-alkoxy, nitro, halogen, phenoxy, or mono- or di-halogen-C1-4-alkyl; terpenyl;
tri(C-4-alkyl)silylethyl; heterocyclyl;
heterocyclyl-C1-4-alkyl; or heterocyclyl-C1-4-alkylcarbonyl;

a group of the formula II or IIa R30-(CH2)q-S(O)m-CH2CH2- II
[R30-(CH2)q-S(O)m-CH2]2CH-CH2- IIa wherein R30 is H, carboxy, C1-4-alkoxycarbonyl, hydroxy, amino, or a matrix MA, q is an integer from 1 to 24, and m is 0 or 2; or a group of the formula III or IIIa Phe-S(O)m-CH2CH2- III
[Phe-S(O)m-CH2]2CH-CH2- IIIa wherein m is as defined above, and each Phe is phenyl optionally substituted with hydroxy, amino, C1-4-alkyl, C1-4-alkoxy, nitro, halogen, phenoxy or mono- or di-halogen C1-4-alkyl; or phenyl-C1-4-alkyl optionally monosubstituted in the phenyl moiety with hydroxy, amino, C1-4-alkyl, C1-4-alkoxy, nitro, halogen, phenoxy, or mono- or di-halogen-C1-4-alkyl;

a group of the formula IV

R40CH2CH(CH2R50)CH2- IV

wherein R40 and R50 independently are halogen; or a group Q-(Spacer)r-, where r is an integer 0 or 1 and Q
is a matrix MA or a group -COO-MA;

for the preparation of a pharmaceutical composition for the treatment or prophylaxis in humans of conditions involving infection by Helicobater pylori of human gastric mucosa.

2. Use according to claim 1 in which Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 are 0.

3. Use according to claim 1 or 2 in which at the most four, preferably at the most three, in particular one or two of R1A, R2A, R3A, R4A, R1B, R2B, R3B, R4B, R1C, R2C, R3C, R4C, R1D, R2D, R3D, R4D, R1E, R2E, R3E or R4E is a group of formula VII.

4. Use according to any of claims 1-3 in which R1A is a group VII
in the .alpha.-configuration.

5. Use according to any of claims 1-3 in which the configuration of R1A, R2A, R3A and R4ACH2 in A are D-galacto, A being in the .beta.-configuration.

6. Use according to any of claims 1-3 in which R1A is a group VII
in the .alpha.-configuration and the configuration of R1A, R2A, R3A and R4ACH2 in A are D-galacto, A being in the .beta.-configuration.

7. Use according to any of claims 1-3 in which R2B is Z3, Z5, Z8, or Z12, and the configuration of R1B, R2B, R3B, and R4BCH2 in B
are D-gluco, B being in the .beta.-configuration.

8. Use according to any of claims 1-3 in which R1B is an acetamido group.

9. Use according to any of claims 1-3 in which R1A is a group VII
in the .alpha.-configuration; the configuration of R1A, R2A, R3A and R4ACH2 in A are D-galacto, A being in the .beta.-configuration; R2B is Z3, Z5, Z8, or Z12; and the configuration of R1B, R2B, R3B, and R4BCH2 in B are D-gluco, B being in the .beta.-configuration and R1B
is an acetamido group.

10. Use according to any of claims 1-9 in which R3B is a group of the formula VII in the .alpha.-configuration.

11. Use according to any of claims 1-10 in which the Configurations of R1A, R2A, R3A, and R4ACH2 in A and of R1B, R2B, R3B, and R4BCH2 in B are D-galacto, and the configurations of R1C, R2C, R3c, and R4cCH2 in C are D-gluco, A being in the .alpha.-configuration, and B and C being in the .beta.-configuration, and in which R1B and R3c are groups of the formula VII in the .alpha.-configuration, and in which R1A and R1C are acetamido groups, and R2B is Z5, Z8 or Z12, and R2C is Z6, Z9 or Z13,

12. Use according to claim 6 in which A is Fuc.alpha.1-2Gal.beta..

13. Use according to claim 9 in which A-Z3-B is Fuc.alpha.1-2Gal.beta.1-3GlcNAc.beta.; or Fuc.alpha.1-2Gal.beta.1-3(Fucal-4)GlcNAc.beta..

14. Use according to claim 9 in which A-Z5-B-Z6-C is Fuc.alpha.1-2Gal.beta.1-3GlcNAc.beta.1-3Gal.beta.; or Fuc.alpha.1-2Gal.beta.1-3(Fucal-4)GlcNAc.beta.1-3Gal.beta..

15. Use according to claims 9 or 11 in which A-Z8-B-Z9-C-Z10-D is GalNAc.alpha.1-3(Fuc.alpha.1-2)Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.; or Fuc.alpha.1-2Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4Glc.beta..

16. Use according to claim 11 in which A-Z12-B-Z13-C-Z14-D-Z15-E
is GalNAc.alpha.1-3(Fuc.alpha.1-2)Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4Glc.beta..

17. Use according to any of claims 1-16 in which R is a group Q-(Spacer)r-, where r is an integer 0 or 1 and Q is a matrix MA.

18. Use according to any of claims 1-17 in which the Spacer is defined (W)v-S'-P', wherein S' is an C1-24 alkyl, an C2-24 alkenyl, an C1-24alkylaryl, an arylC1-24alkyl an arylC1-24alkylaryl, an C1-24alkylarylC1-24alkyl group which groups may be interrupted by carbonyl, thiocarbonyl, oxycarbonyl, carbonyloxy, carbonylamino, aminocarbonyl, aza, oxa or thia groups; an aryl group, an aryloxy, an C1-24alkoxy, a polyethyleneglycol group, a steroid group, a sphingoid group; all groups may be substituted with carboxyl, C1-4alkylcarbonyl, amide, hydroxy, alkoxy, aryloxy, phenoxy;

P' is NH-C(S), NH-C(O), C(O), NH, C(S), C(O)O, (O)CO, SO, SO2, SO3, SO4, PO3, PO4;
W is NH C(S), NH-C(O), C(O), C(S), C(O)O, (O)CO, SO, SO2, SO3, SO4, PO2, PO3, PO4, with the proviso that when Z1, Z2, Z4, Z7, Z11 or Z16 are CH2 then W cannot: be PO2, with the proviso that when Z1, Z2, Z4, Z7, Z11 or Z16 are O or S
then W cannot be (O)CO, SO4 or PO4, and with the proviso that when Z1, Z2, Z4, Z7, Z11 or Z16 are NH then W cannot be NH-C(S), NH-C(O), (O)CO, SO4, PO4; and v is an integer 0 or 1.

19. Use according to any of claim 18 in which the spacer is selected from spacer 1 spacer 2 spacer 3 spacer 5 spacer 4

20. Use according to claims 1-17 in which MA is HSA, BSA or PAA.

21. Use according to claim 1 in which the compound is selected from [Fuc.alpha.l-2Gal.beta.1-Spacer]n-MA;
[Fuc.alpha.l-2Gal.beta.1-3GlcNAc.beta.l-Spacer]n-MA;
[Fuc.alpha.l-2Gal.beta.1-3(Fuc.alpha.l-4)GlcNAc.beta.1-Spacer]n-MA;
[Fuc.alpha.l-2Gal.beta.1-3GlcNAc.beta.l-3Gal.beta.1-Spacer]n-MA;
[Fuc.alpha.l-2Gal.beta.l-3(Fucal-4)GlcNAc.beta.1-3Gal.beta.1-Spacer]n-MA;
[GalNAc.alpha.1-3(Fuc.alpha.l-2)Gal.beta.l-3(Fuc.alpha.l-4)GlcNAc.beta.1-3Gal.beta.1-Spacer]n-MA;
[Fuc.alpha.l-2Gal.beta.1-3(Fuc.alpha.l-4)GlcNAc.beta.1-3Gal.beta.1-4Glc.beta.1-NH]nMA;
[GalNAc.alpha.l-3(Fucal-2)Gal.beta.l-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4Glc.beta.1-Spacer]n-MA;
wherein the Spacer is selected from the group defined in claim 19, n is an integer 1-40 when MA is HSA or BSA, and n is an interger 10-10000 when MA is PAA.

22. Use according to claim 1 in which the compound is selected from [Fuc.alpha.1-2Gal.beta.1-Spacer 1]n-HSA;
[Fuc.alpha.1-2Gal.beta.1-Spacer 2]n-PAA;
[Fuc.alpha.1-2Gal.beta.1-Spacer 4]n-HSA;
[Fuc.alpha.1-2Gal.beta.1-Spacer 5]n-PAA;
[Fuc.alpha.1-2Gal.beta.1-3GlcNAc.beta.1-Spacer 5]n-PAA;
[Fuc.alpha.1-2Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-Spacer 5]n-PAA;
[Fuc.alpha.l-2Gal.beta.1-3GlcNAc.beta.1-3Gal.beta.1-Spacer 3]n-HSA;
[Fuc.alpha.l-2Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-Spacer 3]n-HSA;
[Fuc.alpha.l-2Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-4Glc.beta.1-NH]n-PAA;
[GalNAc.alpha.1-3(Fuc.alpha.l-2)Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-3Gal.beta.1-Spacer 3]n--HSA;
wherein Spacer 1, Spacer 2, Spacer 3, Spacer 4 and Spacer 5 are defined as in claim 19 and n is an integer 1-40 when MA is HSA, and n is an interger 10-10000 when MA is PAA.

23. Use according to any of claims 1-22 wherein the compound of formula Ia, Ib, Ic, Id, Ie or If is adapted to be administered in combination with a preparation for standard therapy of gastritis or ulcus, especially preparations containing omeprazole, cimetidine, ranitidine, lansoprazole, pantoprazole, sucralfate, famotidine, nizetidine, magnesium hydroxide, aluminium hydroxide, calcium carbonate, simethicone or magaldrate.

24. Use according to any of claims 1-23 wherein the compound of formula Ia, Ib, Ic, Id, Ie or If is adapted to be administered in combination with a preparation for a course of therapy with an antimicrobial agent, especially preparations containing:

.beta.-lactam antibiotics such as amoxicillin, ampicillin, cephalothin, cefaclor or cefixime; or macrolides such as erythromycin, or clarithromycin; or tetracyclines such as tetracycline or doxycycline; or aminoglycosides such as gentamycin, kanamycin or amikacin; or quinolones such as norfloxacin, ciprofloxacin or enoxacin; or others such as metronidazole, nitrofurantoin or chloramphenicol;
or preparations containing bismuth salts such as bismuth subcitrate, bismuth subsalicylate, bismuth subcarbonate, bismuth subnitrate or bismuth subgallate.

25. A method of treating and/or preventing diseases in humans caused by infection by Helicobacter pylori of human gastric mucosa, said method comprising administering to a patient in need thereof an effective amount of a compound of the formula Ia, Ib, Ic, Id, Ie or If as defined in claims 1-22.

26. A method of treating and/or preventing diseases in humans caused by infection by Helicobacter pylori of human gastric mucosa, said method comprising administering to a patient in need thereof an effective amount of a compound of the formula Ia, Ib, Ic, Id, Ie of If as defined in claims 1-22 in combination with at least one anti-ulcer or anti-gastritis medicament, or with at least one antimicrobial agent, or with mixtures thereof.

27. A pharmaceutical composition comprising a compound of the formula Ia, Ib, Ic Id, Ie or If as defined in claims 1-22 or a mixture of such compounds, in combination with at least one anti-ulcer or anti-gastritis medicament, or with at least one antimicrobial agent, or with mixtures thereof, and with a pharmaceutically acceptable carrier.

28. A pharmaceutical composition according to claim 27 in which the anti-ulcer or anti-gastritis medicament is selected from a gastric secretion inhibiting compound and an antacid.

29. A pharmaceutical composition according to claim 28 in which the gastric secretion inhibiting compound is selected from cimetidine, ranitidine, famotidine, nizatidine, omeprazole, lansoprazole, pantoprazole, and sucralfate.

30. A pharmaceutical composition according to claim 28 in which the antacid is selected from Al(OH)3, Mg(OH)2, CaCO3, Na2CO3, NaHCO3, aluminium magnesium hydroxide or its hydrate, simethicone.

31. A pharmaceutical composition according to claim 27 in which the antimicrobial agent is selected from .beta.-lactam antibiotics such as amoxicillin, ampicillin, cephalothin, cefaclor or cefixime; macrolides such as erythromycin or clarithromycin;
tetracyclines such as tetracycline or doxycycline;
aminoglycosides such as gentamycin, kanamycin or amikacin;
quinolones such as norfloxacin, ciprofloxacin or enoxacin;
bismuth salts such as bismuth subcitrate, bismuth subsalicylate, bismuth subcarbonate, bismuth subnitrate or bismuth subgallate;
heterocyclic antibiotics such as metronidazole or nitrofurantoin; and benzene derivatives such as chloramphenicol.

32. Novel compound [Fuc.alpha.1-2Gal.beta.1-Spacer 1]n-HSA, wherein Spacer 1 is and n is an integer 1-40.

33. Novel compound [Fuc.alpha.1-2Gal.beta.1-Spacer 2]n-PAA, wherein Spacer 2 is and n is an integer 10-10000.

34. Novel compound [Fuc.alpha.1-2Gal.beta.1-Spacer 4]n-HSA, wherein Spacer 4 is and n is an integer 1-40.

35. Novel compound [Fuc.alpha.1-2Gal.beta.1-Spacer 5]n-PAA, wherein Spacer 5 is and n is an integer 10-10000.

36. Novel compound [Fuc.alpha.1-2Gal.beta.1-3GlcNAc.beta.1-spacer 5]n-PAA, wherein Spacer 5 and n are as defined in claim 35.

37. Novel compound [Fuc.alpha.1-2Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc.beta.1-Spacer 5]n--PAA, wherein Spacer 5 and n are as defined in claim 35.

38. Novel compounds according to any of claims 32-37 wherein the compound is adapted to be administered in combination with a preparation for standard therapy of gastritis or ulcus, especially preparations containing omeprazole, cimetidine, ranitidine, lansoprazole, pantoprazole, sucralfate, famotidine, nizetidine, magnesium hydroxide, aluminium hydroxide, calcium carbonate, simethicone or magaldrate.

39. Novel compounds according to any of claims 32-37 wherein the compound is adapted to be administered in combination with a preparation for a course of therapy with an antimicrobial agent, especially preparations containing:

.beta.-lactam antibiotics such as amoxicillin, ampicillin, cephalothin, cefaclor or cefixime; or macrolides such as erythromycin, or clarithromycin; or tetracyclines such as tetracycline or doxycycline; or aminoglycosides such as gentamycin, kanamycin or amikacin; or quinolones such as norfloxacin, ciprofloxacin or enoxacin; or others such as metronidazole, nitrofurantoin or chloramphenicol;
or preparations containing bismuth salts such as bismuth subcitrate, bismuth subsalicylate, bismuth subcarbonate, bismuth subnitrate or bismuth subgallate.

40. Novel compounds according to any of claims 32-37 for use in therapy.

41. A process for the preparation of novel compounds as defined in any of claims 32-37 by methods known in the art.

42. A process for the preparation of the novel compounds of formula Ia, Ib, Ic, Id, Ie and If, as defined in claim 1, which process comprises i) conversion of a monosaccharide to a glycoside with an aglycon Ra to form the Ra-glycoside derivative in such a way that the Ra-glycoside is possible to transform to a glycosyl donator by activation at the anomeric centre, ii) followed by subsequent derivatisation of new functional groups Rb, iii) condensation of the Ra-glycosides substituted with Rb, if several glycosidic linkages are desired this step iii) will be repeated, iv) followed by introducing the substituent group Rc defined as (Z1-Z16)-R at the reducing end, and if necessary removal of protective groups.