WO2003018598A2 - Monosaccharide derivatives - Google Patents

Abstract

Monosaccharide (pyranoside) conjugates of the formula have been found to be useful as enhancers and/or inhibitors of heparin binding to FGF. The compounds have the potential to be useful in regenerative medicine or for treatment of pathological disease associated with FGF activity or as glycoprocessing inhibitors. In particular the compounds are potential modulators of fibroblast growth factors (FGFs) and fibronectin, as mitogenic agents and as inhibitors of endothelial cell survival.

Description

"Monosaccharide derivatives"

The invention relates to monosaccharide (pyranoside) conjugates and their therapeutic use, in particular their use as potential modulators of fibroblast growth factors (FGFs) and fibronectin, as mitogenic agents and as inhibitors of endothelial cell survival.

Carbohydrate recognition plays an extremely important role in many physiologically significant processes. Some examples are in cell-cell interactions such as (i) microbial and viral recognition of host cell surface proteins; (ii) antigen antibody recognition; (iii) cell adhesion and (iv) extracellular signalling processes. Molecules that alter these biological processes are of considerable interest in the development of therapeutics for treatment of disease. Some carbohydrate based therapeutics that have found use in the clinic include heparin (anticoagulant), relenza and tamiflu (anti- influenza agents), miglitol and acarbose (diabetes).

Glycosaminoglycans (GAGs) are one family of the important carbohydrates that are linear sulfate substituted polymers composed of disaccharide repeating units (e.g. heparin and heparan sulfate). They mediate numerous physiological processes including cell adhesion, activation of growth factors, blood coagulation, lipid metabolism and infection (Lander, Chem. Biol. (1994), 1:73-78). Drugs that promote or inhibit binding of GAGs to proteins will be useful medicaments for treating a variety of diseases. Despite their promising potential, GAGs bind to many proteins and at present cannot be used as specific agonists or antagonists for any one protein. Work has been published on the synthesis and identification of the oligosaccharide sequences that are required for biological activity including identification of the unique pentasaccharide sequence that binds to antithrombin (Linhardt and Toida in Carbohydrates in Drug Design (Edited by Z. J. Witzak, K. A. Nieforth, Marcel Dekker, New York, 1997): 277-342) and the synthesis of a hexadecasaccharide which has the full anticoagulation properties of heparin with no side effects (Petitou et al., Angew. Chem. Int. Ed. Engl., (1998) 37: 3009). Despite these successes, there are problems in using this type of synthetic oligosaccharide as a drug as its large scale synthesis is extremely difficult and expensive and is not attractive for the pharmaceutical industry due to the structural complexity and number of steps involved. They are also hydrophilic and have poor bioavailability undermining their potential in drug development (Wong, Accounts of Chemical Research (1999) 32: 367-85).

Fibroblast Growth Factors (FGFs) have important roles in a variety of biological processes such as cell growth, differentiation, angiogenesis

(formation of blood vessels during wound repair and in tumours) and wound healing. They are implicated in a number of pathological processes such as diabetic retinopathy, rheumatoid disease and tumour growth, which makes them prime targets for the development of inhibitors to treat these diseases. Also promoters of growth factor activity have potential roles in regenerative medicine (Petit-Zeman, Nature Biotechnology (2001), 19: 201-

206). The cellular receptors for FGFs are receptor tyrosine kinases. These receptors are activated by ligand induced dimerisation. An additional feature is that high affinity binding of FGF to its receptor requires heparin or heparan sulfate as a co-factor. Crystallographic studies have revealed that for FGF-2 the ligand receptor complex consists of two molecules of receptor and two molecules of FGF and binding sites have been identified for oligosaccharides. There is growing experimental evidence which suggests that these oligosaccharides spatially organize the ligands to functionally associate with the receptor and that they also have significant interactions with both ligand and receptor in the biologically active complex (Stauber et al, Proc. Natl. Acad. Sci. U. S. A. (2000), 97: 49-54; Plotnikov et al., Cell (2000),

101: 413-24; Plotnikov et. al., Cell (1999), 98:641-50; DiGabriele et al, Nature (2000), 393: 812-817; Pellegrini et al, (2000), 407: 1029-1034; Schlessinger, et al, Molecular Cell (2000), 6: 743). The eventual consequence of exposing cells to growth factors can be cell movement, differentiation, proliferation or protection from death. There is some evidence that growth factors and their receptors may be useful as targets of anti-cancer (breast cancer) therapy as there is recent evidence that they can induce apoptosis or increase the sensitivity of cells for chemo- or hormonal therapy induced apoptosis (de Jong et al, Breast Cancer Research and Treatment (2001), 66: 201-208; Liekens et al, Cancer Research (2001), 61: 5057-5064). In some cases FGF can inhibit apoptopic pathways (Kondo et al, FASEB Journal (1996), 10: 1192-97).

The development of compounds with pro-angiogenic and anti-angiogenic properties has been of interest recently and growth factors and their receptors are considered important targets (Cristofanilli et al, Nature

Reviews Drug Discovery (2002), 1: 415-260). What is of particular interest for angiogenic therapy is novel compounds that would promote or inhibit endothelial cell proliferation/ survival pathways. FGF-2 is released by endothelial cells, which drives proliferation of these cells and potently suppresses apoptopic cell death. Evidence has been published that shows that FGF-2 activity is inhibited by a neutralising anti-FGF-2 antibody and this leads to increased apoptosis (Garcia and D'Amore, Invest. Opthalmol. Vis. Sci. (1999), 40: 2945).

Work by Ornitz et al, (1995), Science, 268: 432-36, has shown that synthetic di- and tri-saccharides can bind to FGF-2 and FGF-1 and activate the FGF receptor. Most of the active di- and trisaccharides exhibited ability to induce cell growth (mitogenesis) and to inhibit heparin binding to the FGFs as well as enhancing binding of FGF to the receptors. Several of the oligosaccharide fragments were non-sulfated unlike heparin and yet retained biological activity and were considerably smaller than the heparin oligosaccharides

(octasaccharides) known to activate FGFs. Although the binding affinity was weaker than heparin, the mitogenic activity was promising in some cases. The differences in activity of the individual saccharides indicated highly specific interactions with their target proteins. The 3-dimensional structure of the protein (FGF-2) complexed with a disaccharide and a trisaccharide showed occupation of the heparin binding site by the sugars and occupation of another site called site 2' (Ornitz et al., Science (1995), 268: 432-36). Site 20 as called by Ornitz et al, has proven to be physiologically relevant in separate work by Moy et al, (1997), Biochemistry, 36: 4782-91). More recent crystallographic work suggests an alternative mechanism by which small di- and trisaccharides could activate the FGF receptor in the presence of FGF (Schessinger et al (2000), Molecular Cell, 6:743).

The development of inhibitors of heparin binding to growth factors (FGF and Vascular Endothelial Growth Factor, NEGF) has received some attention recently. Zhang et al., Bioorg. Med. Chem. (2001), 9: 825-36, identified inhibitors from combinatorial libraries of Ugi four component condensation reactions. These compounds are generally non-carbohydrate dimers that contain charged acid or sulfonate groups and these compounds are in the molecular weight range >700.

It is clear that any efficient modulator (promoter or inhibitor) of (fibroblast) growth factors or of endothelial cell proliferation or survival would have a very beneficial impact therapeutically. A simple compound that could replace oligosaccharides as drugs would also be beneficial.

Fibronectin is an extracellular matrix component with important roles in cell adhesion and inflammation, wound healing, migration of cells during embryonic development and cancer metastasis. It contains both fibronectin receptor and heparin binding sites. The heparin binding site is implicated in adhesion during metastasis (Heavner, Drug Discovery Today (1996), 1: 295- 304). Fibronectins are ligands for the integrin family of adhesion receptors.

These molecules function as signalling molecules and cell adhesion is often coupled to signal transduction in cells. There is interest in the development of novel antagonists of the fibronectin and fibronectin receptor interaction due to their potential in cancer therapy and other diseases by inhibition of cell adhesion and signal transduction pathways.

Statements of Invention

According to the invention there is provided a compound of the Formula

wherein

A- to A is any one or more of the same or different of

OH; F; or NH_2;

wherein when

B is CO₂H,

X is NR²CO; NR²COCH₂; NR²COCH₂O; NR²COCH=CH;

NR²COCH₂CH₂; NR²COCH₂CH₂CO; NR²SO₂CH₂, NR²SO₂CH₂O;

NR²SO₂CH=CH; NR²SO₂CH₂CH_2; NR²SO₂CH₂CH₂CO; CONR²; CONR²CH₂; CONR²CH₂O; CONR²CH=CH; CONR²CH₂CH₂;

CONR²CH₂CH₂CO; SO₂NR²CH₂; SO₂NR²CH₂O; SO₂NR²CH=CH; SO₂NR²CH₂CH₂; or SO₂NR²CH₂CH₂CO;

wherein R² is H or alkyl

and R is benzene; pyridine; pyrazine; thiophene; furan; cyclopropyl; indole; quinoline; naphthalene; chrom-4-enone;

or tetrahydrofuran,

which may be unsubstituted or substituted with any one or more or different of halogen; trifluoromethyl; OMe; Me; NO₂; phenyl; CONH-sugar or NHCO-sugar, or

X is O; S; SO₂; OCH₂CH₂; SCH₂CH₂; SO₂CH₂CH₂; OCH₂; SCH₂ or

and

which may be unsubstituted or substituted with any one or more or different of halogen; OMe; Me; NO₂; trifluoromethyl or OH,

or when

B is CH2OH

X is NR²CO; NR²COCH₂; NR²COCH₂O; NR²COC₆H₄;

NR²COCH=CH; NR²COCH₂CH₂CO; NR²SO₂CH₂; NR²SO₂CH₂0;

NR²SO₂CH=CH; NR²SO₂CH₂CH₂CO; CONR²; CONR²CH₂; CONR²CH₂O; CONR²CH=CH; CONR²CH₂CH₂CO; SO₂NR²CH₂;

SO₂NR²CH₂O; SO₂NR²CH=CH; or SO₂NR²CH₂CH₂CO;

wherein R² is H or alkyl,

and R is difluorobenzene; dichlorobenzene; chlorofluorobenzene; dimethylbenzene; trifluoromethylbenzene; trimethoxybenzene; phenylbenzene; pyrazine; thiophene; furan; cyclopropyl; indole; quinoline; CONH-sugar; NHCO-sugar; or

-\\ f

or R is pyridine, pyrazine, thiophene, furan, cyclopropyl, indole and quinoline containing at least one ring substituent selected from any one or more or different of halogen; trifluoromethyl; OMe; Me; NO₂; or phenyl,

and epimers and pharmacologically acceptable salts, esters, amides and prodrugs thereof.

The invention also provides a compound of the Formula

wherein

A] to A is any one or more of the same or different of OH; F or NH_2;

wherein when

B is CO₂H

X is NR²CO; NR²COCH₂, NR²COCH₂O; NR²COCH=CH; NR²COCH₂CH₂; NR²COCH₂CH₂CO; NR²SO₂CH₂; NR²SO₂CH₂O;

NR²SO₂CH=CH; NR²SO₂CH₂CH_2; NR²SO₂CH₂CH₂CO; CONR²; CONR²CH₂; CONR²CH₂0; CONR²CH=CH; CONR²CH₂CH₂; CONR²CH₂CH₂CO; SO₂NR²CH₂; SO₂NR²CH₂O; SO₂NR²CH=CH; SO₂NR²CH₂CH₂ or SO₂NR²CH₂CH₂CO;

wherein R² is H or alkyl

R is benzene; pyridine; pyrazine; thiophene; furan; cyclopropyl; indole; quinoline; naphthalene; chrom-4-enone;

X < . v< or tetrahydrofuran,

which may be unsubstituted or substituted with any one or more or different of halogen; trifluoromethyl; OMe; Me; NO₂; phenyl; CONH-sugar or NHCO-sugar;

and epimers and pharmacologically acceptable salts, esters, amides and prodrugs thereof.

The invention also provides a compound of the Formula

wherein

Ai to A₃ is any one or more of the same or different of OH; F or NH_2;

wherein B is CO₂H

X is O, S, SO₂; OCH₂CH₂;, SCH₂CH₂; SO₂CH₂CH₂;, OCH₂; SCH₂ or SO₂CH₂, and

which may be unsubstituted or substituted with any one or more or different of halogen; OMe; Me; NO₂ or OH,

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

The invention also provides a compound of the Formula

wherein

Ai to A is any one or more of the same or different of OH; F; or NH_2;

B is CH₂OH X is NR²CO; NR²COCH₂; NR²COCH₂O; NR²COCH=CH NR²COCH₂CH₂CO; NR²SO₂CH₂; NR²SO₂CH₂O; NR²SO₂CH=CH NR²SO2CH₂CH₂CO; CONR²; CONR²CH₂; CONR²CH₂O; CONR²CH=CH CONR²CH₂CH₂CO; SO₂NR²CH₂; SO₂NR²CH₂O; SO₂NR²CH=CH or SO₂NR²CH₂CH₂CO;

wherein R² is H or alkyl,

and R is difluorobenzene; dichlorobenzene; chlorofluorobenzene; dimethylbenzene; trifluoromethylbenzene; trimethoxybenzene; phenylbenzene; pyrazine; thiophene; furan; cyclopropyl; indole; quinoline; CONH-sugar; NHCO-sugar; or

-V J

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

The invention also provides a compound of the formula

wherein

Ai to A₃ is any one or more of the same or different of OH; F or NH_2;

B is CH₂OH X is NR'CO; NR'COCH₂; NR OCH₂O; NR COCH=CH

NR²COCH₂CH₂CO; NR²SO₂CH₂; NR²SO₂CH₂O; NR²SO₂CH=CH NR²SO₂CH₂CH₂CO; CONR²; CONR²CH₂; CONR²CH₂O; CONR²CH=CH CONR²CH₂CH₂CO; SO₂NR²CH₂; SO₂NR²CH₂O; SO₂NR²CH=CH or SO₂NR²CH₂CH₂CO; wherein R² is H or alkyl

or

R is pyridine, pyrazine, thiophene, furan, cyclopropyl, indole and quinoline which containing at least one ring substituent selected from any one or more or different of halogen; trifluoromethyl; OMe; Me; NO₂ or phenyl,

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

The invention also provides a compound of the formula

wherein

B is CH₂OH

X is NR²CO; NR²COCH₂; NR²COCH₂0; NR²COCH=CH

NR²COCH₂CH₂CO; NR²SO₂CH₂; NR²SO₂CH₂O; NR²SO₂CH=CH NR²S0₂CH₂CH₂CO; CONR²; CONR²CH₂; CONR²CH₂O; CONR²CH=CH CONR²CH₂CH₂CO; SO₂NR²CH₂; SO₂NR²CH₂O; SO₂NR²CH=CH; or SO₂NR²CH₂CH₂CO, and

R is thiophene, unsubstituted or substituted with one or more or different of halogen; trifluoromethyl; OMe; Me; NO₂; phenyl; CONH-sugar or CONH- sugar,

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

Preferably the halogen is selected from any one or more of F, Cl, Br or I.

Preferably the sugar is selected from any one or more of glucose, galactose, mannose, glucuronic acid or iduronic acid.

Preferably the R group is mono, di or tri-substituted.

One embodiment of the invention provides a compound of the Formula

wherein

Ai to A₃ is any one or more of the same or different of H; OH; F or NHAc,

X is NHCO or NHCOCH₂O,

and R is an aromatic or heteroaromatic group, which may be unsubstituted or substituted or a group selected from any one or more of

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

Preferably Aj to A₃ are each hydroxy. Preferably the aromatic or hetero-aromatic group is selected from any one of benzene; pyridine; thiophene; pyrazine or indole.

Most preferably the aromatic or heteroaromatic group is substituted with any one or more or different of halogen;trifluoromethyl; OMe; Me; NO₂; phenyl; CONH-sugar or NHCO-sugar.

Most preferably the aromatic or heteroaromatic group is substituted with fluorine or chlorine.

Compounds of this structure were shown to be potent potent inhibitors of binding to fibronectin.

The invention preferably provides a compound wherein X is OC2H₄ and R is a phthalimide.

The invention also provides a compound wherein X is HNCO and R is benzene. Compounds of this structure are monosaccharides whereas the only compounds, which have shown activity in FGF based assays previously are oligosaccharides. Oligosaccharides are more difficult and expensive to prepare and may have poor bioavailability. The compounds described herein require less synthesis steps and have improved pharmacokinetics and better drug properties than oligosaccharides as they have reduced numbers of hydrogen bond acceptors and donors.

Compounds wherein X is OC₂H₄ and R is succinimide andwherein X is NHCO and R is an aromatic group such as thiophene, 4-pyridine or difluorobenzene have in particular been shown to inhibit FGF binding to heparin. The inhibitors have been shown to inhibit the survival ability of endothelial cells. Survival of these cells is driven by FGF-2 and the activity observed is consistent with compounds inhibiting the FGF-2:heparan sulfate proteoglycan:FGF receptor interaction.

The invention also provides a compound of the Formula

wherein

Ai to A₃ is any one or more of the same or different of H; OH; F or NHAc;

X is NHCO; OCH₂CH₂; NHCOCH2; NHCOCH2CH2CO or NHCOCH₂O

and R is selected from any one or more of

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

Preferably Ai to A₃ are each hydroxy. Such compounds were shown in particular to be inhibitors of FGF binding to heparin-albumin.

The invention further provides a compound of the Formula

C 300₂₂HH HO- ^" HO)Λ- ^- - _R

HO

wherein

X is O or OCH₂CH₂, or

and

R is selected from any one or more of

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

Such compounds were shown in particular to be stimulators of FGF binding to heparin-albumin.

The invention also provides a compound of the Formula

wherein

B is CO₂H or CH₂OH

X is NHCOCH₂O or NHCO,

and

R is selected from any one or more of

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

Such compounds were shown in particular to be inhibitors of endothelial cell binding.

The invention also provides a compound of formula

wherein

Ai to A₃ is OH; B is CO₂H; X is OCr alkyl; and R is a cycloalkanone,

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

The invention further provides a compound of the formula

wherein A, to A₃ is OH;

B is CO₂H; and X is NR³CO; NR³COd.₆alkenyl; NR³COCι_-6 alkyl; NR³CO;

NR³COCH₂O;

wherein R is H, alkyl, alkenyl or alkynyl, and

R is H, aromatic group or cycloalkyl group which may be substituted or unsubstituted,

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

The invention further provides a compound selected from the following

(N-(Benzoyl)-2,3,4-tri-O-acetyl- ^D-glucopyranosylamine)uronic acid, methyl ester;

Succinimidoethyl-2,3,4-tri-0-acetyl-β-D-glucopyranuronic acid, methyl ester;

Phthalimidoemyl-2,3,4-tri-O-acetyl-β-D-glucopyranuronic acid, methyl ester;

(l,4-Dioxaspirol[4,5]dec-2-yl)-2,3,4-tri-Ο-acetyl-β-D-glucopyranuronic acid, methyl ester; (3-Benzoylphenyl)-2,3,4-tri-0-acetyl-β-D-glucopyranuronic acid, methyl ester;

N-(2-Biphenylcarbonyl)-2,3,4-tri-0-acetyl- ?-D-glucopyranosylamine) uronic acid, methyl ester;

(N-(2-Phenylquinoline-4-carbonyl)-2,3,4-tri-0-acetyl-y5-D-glucopyranosyl amine) uronic acid, methyl ester; (N-(4-Chlorophenylacetyl)-2,3,4-tri-O-acetyl-β-D-glucopyranosylamine) uronic acid, methyl ester;

(N-(3-methyl-2-but-2-enoly)-2,3,4-tri-O-acetyl-β-D-glucopyranosylamine) uronic acid, methyl ester; (N-(3-furan-2-yl-acryloyl)-2,3,4-tri-O-acetyl-β-D-glucopyranosylamine) uronic acid, methyl ester;

(N-(4-Biphenylacetyl)-2,3,4-tri-0-acetyl-β-D-glucopyranosylamine)uronic acid, methyl ester; (N-(2-Methylpentanoyl)-2,3,4-tri-O-acetyl-β-D-glucopyranosylamine) uronic acid, methyl ester;

(N-2-cyclopropanecarbonyl)-2,3,4-tri-0-acetyl-β-D-glucopyranosylamine) uronic acid, methyl ester;

(N-(2,4-dichlorophenoxyacetyl)-2,3,4-tri-O-acetyl-β-D-glucopyranosyl- amine)uronic acid, methyl ester;

(N-(2-Pyrazinoyl)-2,3,4-tri-0-acetyl- MXglucopyranosyIamine)uronic acid, methyl ester;

(N-(2-Thiophenoyl)-2,3,4-tri-O-acetyl- ^D-glucopyranosylamine)uronic acid, methyl ester; (N-(2-Pyridine-4-carbonyl)-2,3,4-tri-0-acetyl- ?-D-glucopyranosylamine) uronic acid, methyl ester;

(N-(2-Chloro-4-nitrobenzoyl)-2,3,4-tri-O-acetyl-/?-D-glucopyranosyl- amine) uronic acid, methyl ester;

(N-(3,4-DifluorobenzoyI)-2,3,4-tri-O-acetyl- ^D-glucopyranosylamine) uronic acid, methyl ester;

(N-(3-TrifluoromethylbenzoyI)-2,3,4-tri-0-acetyI- 7-D-glucopyranosyl-amine) uronic acid methyl ester;

(N-(3,5-Dimethylbenzoyl)-2,3,4-tri-O-acetyl-_/0-D-glucopyranosylamine) uronic acid, methyl ester; (N-(3,4,5-Trimethoxybenzoyl)-2,3,4-tri-0-acetyl- ?-D-glucopyranosyl- amine)uronic acid, methyl ester;

[N-(naphthalene-2-carbonyl)-2,3,4-tri-O-acetyl- ?-D-glucopyranosylamine] uronic acid, methyl ester; [N-(2-Thiophenoyl)-2,3-di-O-acetyl-4,5-anhydro- 9-D-glucopyranosyl-amine] uronic acid, methyl ester;

[N-(Benzoyl)-2,3-di-O-acetyl-4,5-anhydro- ?-D-glucopyranosylamine] uronic acid, methyl ester; [N-(3,5-Dimethylbenzoyl)-2,3-di-0-acetyl-4,5-anhydro- ?-D-glucopyran- osylamine] uronic acid, methyl ester.

The invention further provides a compound selected from the following

Succinimidoethyl-β-D-glucopyranuronic acid;

Phthalimidoethyl-β-D-glucopyranuronic acid;

[l,4-Dioxaspirol[4,5]dec-2-yl]-β-D-glucopyranuronic acid;

3-Benzoylphenyl-β-D-glucopyranuronic acid;

(Ν-(4-Chlorophenylacetyl)-β-D-glucopyranosylamine)uronic acid; (N-(3-methyl-2-but-2-enoly)-β-D-glucopyranosylamine)uronic acid;

(N- (3-Furan-2-ylacryloy 1) -β-D-glucopyranosylamine) uronic acid;

(N- (2-Methy Ipentanoy 1) -β-D -glucopy ranosy lamine) uronic acid ;

((N-cyclopropanecarbonyl)-β-D-glucopyranosylamine)uronic acid;

(N-((2,4-Dichlorophenoxy)acetyl)-β-D-glucopyranosylamine)uronic acid; (N-(Benzoyl)-_, ?-D-glucopyransylamine)uronic acid;

(N-(3-Trifluoromethylbenzoyl)-/?-D-glucopyranosylamine)uronic acid;

(N-(3,5-Dimethylbenzoyl)- ?-D-glucopyranosylamine)uronic acid;

(N-(3,4,5-Trimethoxybenzoyl)- ?-D-glucopyranosylamine)uronic acid;

(N-(Biphenyl-2-carbonyl)-ff-D-glucopyranosylamine) uronic acid; (N-(2-Phenylquinoline-4-carbony0- ?-D-glucopyranosylamine)uronic acid;

(N-(2-Pyrazinoyl)- ?-D-glucopyranosylarnine)uronic acid;

(N-(2-Thiophenoyl)-/?-D-glucopyranosylamine)uronic acid;

(N-(2-Pyridine-4-carbonyl)-/^D-glucopyranosylamine)uronic acid; (N-(2-Chloro-4-nitrobenzoyl)- 9-D-glucopyranosylamine)uronic acid; [N-(3,4-Difluorobenzoyl)- ?-D-glucopyranosylamine] uronic acid; [ t-(naphthalene-2-carbonyl)- ?-D-glucopyranosylamine] uronic acid; [N-(lH-indole-2-carbonyl)- ^D-glucopyranosylamine] uronic acid; [N(3,5-Dimethylbenzoyl)-anhydro- ?-D-glucopyranosylamine]uronic acid;

(Ν- (4-Oxo-4-phenylbutyryl) -β-D-glucopyranosylamine) uronic acid; (Ν-3 (1 Η-indol-3-yl) -propiony I) -β-D-glucopyranosylamine) uronic acid; (N-(4-biphenylacetyl-2,3,4-tri-O-acetyl-β-D-glucopyranosylamine)uronic acid; (N-3-methyl-4-oxo-2-phenyl-4H-chromene-8-carbonyl-β-D-glucopyran- osyIamine)uronic acid;

(N-(4-Oxo-4-phenyl-butyryl)-β-D-glucopyranosylamine)uronic acid N,N ' -Di- (β-D-galactopyranosy 1) -terephthalamide Thiophene-2-carboxylic acid-N-(β-D-glucopyranosyl)-amide [N-(3,4-Difluorobenzoyl)-/?-D-glucopyranosylamine] uronic acid [N-(naphthalene-2-carbonyl)- ?-D-glucopyranosylamine] uronic acid

[N-(lH-indole-2-carbonyl)- ?-D-glucopyranosylamine] uronic acid (Ν-3(lΗ-indol-3-yl)-propionyl)-β-D-glucopyranosylamine)uronic acid (N-(4-biphenylacetyl-2,3,4-tri-0-acetyl-β-D-glucopyranosylamine) uronic acid N-(3-Methyl-4-oxo-2-phenyl-4H-chromene-8-carbonyl)-β-D- glucopyranosylamine) uronic acid

N,N'-Di(β-D-glucopyranuronosyl)-terephthaIamide (N-(Tetrahydrofuran-2-carbonyI)-/?-D-glucopyranosylamine)uronic acid

The invention also provides a pharmaceutical composition comprising a compound of the invention including a pharmaceutically acceptable carrier or diluent. The invention further provides a pharmaceutical composition comprising a compound of the invention together with FGF and/or heparin for simultaneous and/or separate administration.

The invention further provides use of a compound of the invention for the preparation of a medicament for the treatment and/or prophylaxis of atherosclerosis and human coronary heart disease, chronic myocardial ischemia, rheumatoid arthritis; ulcerative colitis, inflammatory bowel disease, crescentic glomerulonephritis, diabetic retinopathy; retinal ischemia, glomerulosclerosis, age- related macular degeneration; psoriasis, intermittent claudication, bacterial meningitis.

The invention further provides use of a compound of the invention for the preparation of a medicament for the stimulation of bone formation in osteopenic disorders; stimulation of muscle, nerve, cornea (retinal) and colonic and other tissue repair; disease associated with nerve tissue regeneration, e.g. spinal cord injury, multiple sclerosis, Alzheimers disease, Parkinson's disease and for healing of gastric, duodenal, colonic and leg ulcers.

Preferably the compound is used in the preparation of a medicament for use in the modulation of fibronectins or the modulation of fibroblast growth factors.

Preferably the compound is used in the preparation of a medicament for use in the modulation of endothelial cell survival or proliferation, the modulation of glycosaminoglycan binding proteins, most preferably in the modulation of heparin binding proteins. The compounds may be used in the preparation of medicaments for use in the treatment and/or prophylaxis of HIV, bacterial infections, hepatitis infection or diabetes as a result of glycoside inhibition.

The compounds of the invention may also be used in the preparation of a medicament for use in the inhibition of glycosidases or glycosyltransferases or saccharide transport or metabolism in cells.

On embodiment of the invention provides use of a compound of the invention in the preparation of a medicament for the treatment and/or prophylaxis of cancer.

Preferably the cancer is invasive breast cancer, pancreatic cancer, progressive multifocal leukoencephalopathy, Kaposis-sarcoma, prostrate cancer, testicular cancer, endocrine related cancers, ovarian cancer, neuroblastoma, human-malignant mesothelioma, renal cell carcinoma, leukemia, gastric carcinoma, fibromatosis, lung cancer, carcinoma of the bladder, non-Hodgkin's lymphoma, colo-rectal cancer; benign prostatic hyperplasia, venous neointimal hyperplasϊa, intimal hyperplasia.

One embodiment of the invention provides use of a compound of the formula

wherein

X is NHCOCH₂O or NHCO,

and

R is benzene, in the preparation of a medicament for use in the modulation of fibroblast growth factors. Compounds of this structure have been shown to be potent inhibitors of FGF binding to heparin albumin.

Brief description of the Drawings

The invention will be more clearly understood from the following description thereof given by way of example only with reference to the accompanying drawings in which:-

Fig. la is a graph showing the binding of FGF to heparin albumin in presence of heparan sulfate and heparin albumin;

Fig. lb is a graph showing the binding of FGF to heparin albumin in presence of heparin sodium salt;

Fig. 2 is a graph showing the binding of FGF to heparin albumin in the presence of compound of example 1;

Fig. 3 is a graph showing the binding of FGF to heparin albumin in the presence of a compound of example 2;

Fig. 4 is a graph showing the binding of FGF to heparin albumin in the presence of a compound of example 11. Fig. 5 is a graph showing the binding of FGF to heparin albumin in the presence of a compound of example 18

Fig. 6 is a graph showing the binding of FGF to heparin albumin in the presence of a compound of example 19;

Fig. 7 is a graph showing the binding of FGF to heparin albumin in the presence of compounds of examples 17 and 21;

Fig. 8 is a bar graph showing the effect of compounds of examples 2 and 18 and 2 bovine aorta endothelial cell viability;

Fig. 9 is a bar graph showing the effect of compounds of example 18 and 19 on bovine aorta endothelial cell viability;

Fig. 10 is a graph showing the binding of Fibronectin to heparin albumin in the presence of compounds of example 10;

Fig. 11 is a graph showing the binding of Fibronectin to heparin albumin in the presence of compounds of example 21;

Fig. 12 is a graph showing the binding of Fibronectin to heparin albumin in the presence of phenolphthalein-β-D-glucuronide, sodium salt; and

Fig. 13 is a graph showing the effect compounds have on FRIC-11 cell proliferation. Detailed description

The present invention provides compounds, which have been found to be useful as enhancers and inhibitors of heparin binding to FGF. These compounds have the potential to be useful in regenerative medicine or for treatment of pathological disease associated with FGF activity or as glycoprocessing inhibitors. The compounds have the general formula I as shown below. The identification of compounds that enhance binding of (Fibroblast) growth factors to heparin has not been observed previously. Some oligosaccharides and high molecular weight inhibitors of heparin binding to FGF are known. However the inhibition of heparin binding to FGF by simple monosaccharide derivatives has not been described before.

The invention provides compounds of formula I

wherein Ai to A3, B, X and R are as described hereinbefore, and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

Most preferably R is selected from any one or more of succinimide, phthalimide, tetrahydrofuran, dioxaspirol[4,5]dec-2-yl or cyclopropyl, and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof. Combinations of substitu tents and/ or variables resulting in stable compounds are included.

Compounds of formula I may contain one or more asymmetric centres. The invention relates to all possible chiral forms of formula I including mixtures of enantiomers, diastereoisomers.

As used herein, the term 'alkyl' means a straight chain or branched chain group of atoms including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl and the like.

The term alkenyl means a straight chain or branched chain group of at least 2 carbon atoms containing at least one alkene (double) bond including but not limited to ethenyl, propenyl, 1-butenyl, 2-methylpropenyl and the like. The present invention relates to all possible E and Z geometric forms of alkenes.

The term alkynyl means a straight chain or branched chain group of at least 2 carbon atoms and at least one alkyne triple bond including but not limited to ethynyl, propynyl, 1-butynyl, 3-methylbutynyl and the like.

The term aromatic means that at least one unsaturated cyclic ring is present comprising at least 5 atoms. This includes compounds which contain only carbon atoms including but not limited to benzene and naphthalene and the like and also their heterocyclic analogues which contain at least one heteroatom for example N, O, S, NH, including but not limited to pyrrole, thiophene, pyridine, furan, pyrazine, benzofuran, indole, benzothiophene, pyridazine, pyrimidine, pyrazine, isoxazole, oxazole, indazole, quinoline, isoquinoline and the like and includes both substituted and unsubstituted derivatives.

The term cycloalkyl means at least one saturated cyclic ring containing at least 3 carbon atoms including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like and also their heterocyclic analogues where at least one of the atoms is O or N or S. These include but are not limited to oxetane, aziridine, tetrahydrofuran, tetrahydropyran, thietane, azetidine, oxepane, dioxepin, piperidine, azepane, thiepane, dioxolane, dioxane and the like. The cycloalkyl group may be fused with an aromatic group or may contain one or more unsaturated bonds.

The term cycloalkanone means a saturated cyclic ring containing at least 4 atoms where at least one group in the ring is a carbonyl. This includes but is not limited to cyclopentanone, cyclohexanone, cycloheptanone, cyclopentan-

1,3-dione and the like and also their heterocyclic analogues where at least one of the atoms in the ring is not carbon including but not limited to tetrahydrofuran-2-one, tetrahydropyran-2-one, piperidin-2-one, pyrrolidine- 2,5-dione and the like. The cycloalkanone ring may be fused with an aromatic group or contain a double bond and includes but is not limited to indan-1-one, indan-l,3-dione, 3,4-dihydronaphthalen-l-one, isoindole-1,3- dione, pyrrol-2,5-dione, cyclopent-2-en-l,3-dione and the like. The cycloalkanone carbonyl group may be protected as a ketal including but not limited to l,4-dioxa-spiro[4.4]nonane, 1,1-dimethoxycyclopentane, 1,4-dioxa- spiro[4.5]decane and the like. In the case of cycloalkyl and cycloalkanone rings there may be carbon atoms where there can be substitution and this can include geminal substitution and the type of geminal substitution which result in spiro systems.

Substituents on the ring include but are not limited to methyl, phenyl, methoxy, halogen (F, Cl, Br, I), N0₂, NHCO-Sugar, CONH-Sugar, CF₃ or COPh.

The term "pharmaceutically acceptable salts, ester, amides and prodrugs" as used herein refers to carboxylate salts, amino acid addition salts, esters, amides and prodrugs of the compounds of the present invention which are, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, commensurate with a reasonable benefit/ risk ratio, and effective for their intended use. The term also includes the zwitterionic forms, of the compounds of the invention. The term "salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulphate, bisulphate, nitrate, acetate, oxalate, valerate, oleate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, laurylsulphonate salts and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine ethylamine.

Examples of pharmaceutically-acceptable, non-toxic esters of the compounds of this invention include C 1-6 alkyl esters wherein the alkyl group is a straight or branched chain. Acceptable esters also include C5-7 cycloalkyl esters as well as arylalkyl esters such as, but not limited to benzyl. Esters of the compounds of the present invention may be prepared according to conventional methods.

Examples of pharmaceutically-acceptable, non-toxic amides of compounds of this invention include amides derived from ammonia, primary C 1-6 alkyl amines and secondary C 1-6 dialkyl amines, wherein the alkyl groups are straight or branched chain. In the case of secondary amines, the amine may also be in the form of a 5 or 6-membered heterocycle containing one nitrogen atom. Amides of the compounds of the invention may be prepared according to conventional methods.

The term "prodrug" refers to compounds for example esters, that are rapidly transformed in vivo to yield the parent compound for example by hydrolysis in blood or in the cytosol The term is commonly known to those skilled in the art.

Compounds of the invention may be prepared by any suitable method known in the art and/ or by the processes described herein. It will be appreciated that where a particular stereoisomer of formula I is required, the synthetic processes described herein may be used with the appropriate homochiral starting material and/ or isomers may be resolved from mixtures using conventional separation techniques (e.g. HPLC).

The compounds according to the invention may be prepared by the following processes.

In the formulae below, the groups are as defined above unless otherwise indicated. It will be appreciated that in all cases described below that functional groups such as amino, hydroxyl, or carboxyl groups may need to be in protected form before any reaction is initiated. In such instances removal of the protecting group may be the final step of a particular reaction sequence or they may be removed before the final step of the reaction sequence. Suitable protecting groups will be apparent to those skilled in the art.

Compounds of the present invention as depicted by general formula I wherein X contains the group NHCO (e.g. examples 5-33), may be prepared from azides (e.g. intermediate 5) or from amines and their reaction with acids or activated acids as shown for example in Scheme 1 below.

The activated acid may be the acid chloride or another equivalent or the activated acid may be generated in situ from the carboxylic acid by addition of a reagent such as dicyclohexylcarbodiimide (DCC) in the presence of hydroxybenzotriazole (HOBT), N,N-4-dimethylaminopyridine (DMAP) in a solvent such as tetrahydrofuran or using other similar reagents known to those skilled in the art. The reaction of the azide with acids or activated acids is promoted by addition of phosphine reagents including but not limited to triphenylphosphine, tributylphosphine, trimethylphosphine and resin bound phosphines.

Reagents and Conditions: (i) RCOC1, CH₃CN then Ph₃P or diphenylphosphinopolystyrene; (ii) LiOH, H₂0, THF, MeOH; (iii) Pd-C, H_2> -15 °C, THF, 2h; (iv) RC0₂H, DCC, HOBT, DMAP, THF.

Scheme 1

Compounds of the present invention as depicted by general formula I wherein X contains an O or S adjacent to the anomeric centre (for example intermediates 7-33 and examples 1-4) may be prepared from glycosyl donors with suitable acceptors as shown for example in Scheme 2 below. L is any suitable leaving group. In this case it is halogen or imidate but it may also be thioalkyl (SMe, SEt), trifluoromethansulfonate, acetate or any other leaving groups known to those skilled in the art of glycoside synthesis. The reactions may be promoted by addition of base or other activating agents

(heavy metal salts, Lewis acids etc.) known to those skilled in the art. The glycosyldonor may also be 1,6-lactone derivatives which are particularly suitable for the synthesis of the α-glycosides. is

α-glycoside synthesis

Reagents and Conditions: (i) AgC0₃, AgC10₄, mol. sieves, dry CH₂C1_2, RZH (L = Br); (ii) BF₃OEt₂, CH₂C1₂, RZH (L = imidate) (iii) LiOH, H₂0, THF, MeOH; (iii) Pd-C, H₂, -15 °C, THF, 2h. (iv) SnCl₄, TMSOTf, CH₂C1₂ RZH.

Scheme 2

O-Glycosides or S-glycosides may also be prepared by chemical methods similar to those shown in Scheme 3 where an alkoxide or thiolate is generated and reacted with intermediates where L² is a leaving group such as a halogen or trifluoromethanesulfonate.

Scheme 3

Compounds of the present invention as depicted by general formula I wherein X=NH₂C0₂ or NHS0₂R and the like may be prepared from glycosylamines with displacement of a leaving group L² such as the sulf onylchloride (L² = Cl) as shown in Scheme 4 below.

Scheme 4

All the intermediates described above can be purchased or prepared by well known methods.

Intermediates may be obtained in optically pure or racemic form. In the chiral form they provide asymmetric building blocks for enantiospecific synthesis of compounds of general formula I. Any mixtures of final products or intermediates, obtained can be separated on the basis of the physicochemical differences of the constituents, in a known manner, into the pure final products or intermediates, for example by chromatography, distillation, fractional distillation, or by formulation of a salt if appropriate or possible under the circumstances.

Also any compound that is among the carbohydrate derivatives that have been described herein that inhibits the action of glycosidases, glycosyltransferases or glycoprotein processing would have potential in angiogenesis and other diseases such as diabetes, cancer, neurodegenerative disease and for treatment of anti-viral infection and antibacterial infection.

For example some glycosidase inhibitors display interesting activity. Miglitol is in clinical use for treatment of diabetes and N-butyl- deoxynojirimycin (NBJ) is in clinical trials for treatment of neurodegenerative disease. Castanospermine is a known inhibitor of angiogenesis. N-Nonyl deoxynojirimycin is in clinical trials for treatment of hepatitis C viral infection. Glycosyltransferase inhibitors would have similar potential.

The present invention provides a method of treating diseases including invasive breast cancer, pancreatic cancer, progressive multifocal leukoencephalopathy, Kaposis-sarcoma, prostrate cancer, testicular cancer, endocrine related cancers, ovarian cancer, neuroblastoma, human-malignant mesothelioma, renal cell carcinoma, leukemia, gastric carcinoma, fibromatosis, lung cancer, carcinoma of the bladder, non-Hodgkin's lymphoma, colo-rectal cancer; benign prostatic hyperplasia, venous neointimal hyperplasia, intimal hyperplasia, atherosclerosis and human coronary heart disease, chronic myocardial ischemia, rheumatoid arthritis, ulcerative colitis, inflammatory bowel disease, crescentic glomerulonephritis, diabetic retinopathy, retinal ischemia, glomerulosclerosis, age-related macular degeneration, psoriasis, intermittent claudication or bacterial meningitis.

The method may be used also in regenerative medicine and can be used in stimulation of bone formation in osteopenic disorders; stimulation of muscle, nerve, cornea (retinal) and colonic and other tissue repair; disease associated with nerve tissue regeneration, e.g. spinal cord injury, multiple sclerosis, Alzheimers disease, Parkinson's disease and for healing of gastric, duodenal, colonic and leg ulcers. In addition to the above human uses, it is contemplated that these compounds can be used in veterinary uses to treat related diseases.

The method may also be used for treatment of viral infection, bacterial infection, diabetes, neurodegenerative disease and diabetes. In addition the compounds of the invention may have use as and antibacterial or anti- infective.

The pharmaceutical composition containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically- acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

Formulations for oral use may also be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, dispersing or wetting agents. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and/ or one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil or in a mineral oil. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Sweetening, flavouring and colouring agents may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, naturally-occurring phosphatides, and esters or partial esters derived from fatty acids and hexitol anhydrides and condensation products of the said partial esters with ethylene oxide. The emulsions may also contain sweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds of formula I may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. The compounds of formula I may also, if required, be used in conjunction with heparin or other pharmaceutically accepted forms of heparin or heparan sulfate and/ or with pharmaceutically acceptable forms of FGFs. This approach would be suitable where compounds enhance the effect of FGFs and/ or heparin/ heparan sulfate. Similarly compounds may be used in conjunction with heparin or heparan sulfate and/ or fibronectins.

For topical use, creams, ointments, jellies, solutions or suspensions containing the compounds of formula I are employed.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

The following Intermediates and Examples illustrate the preparation of compounds of Formula I, and as such are not intended to limit the invention as set forth in the claims.

General synthesis of the compounds of the invention compounds

(examples): The synthesis N-glucuronamide derivatives was carried out from intermediate 5, which can be prepared easily from glucurono-3,6-lactone (von Roedern et al. J. Am. Chem. Soc. 1996, 118: 10156- 10167). Activation of the azide using triphenylphosphine in the presence of the appropriate acid chloride (method of Boullanger et al, Carbohydr. Res. 2000, 324: 97-106) gave an amide in low to good yields. It was also possible to use the polystyrene supported triphenylphosphine variant for activation of the azide and this simplified the purification procedure in some cases. An amine (intermediate 6) was also prepared by hydrogenation of intermediate 2 and this could be coupled with the appropriate carboxylic acid using standard procedures.

The synthesis of the β-O-glucuronide derivatives was carried out by glycosylation of a glycosyl bromide (intermediate 2) or a trichloroacetimidate (intermediate 4) and subsequent deprotection. A number of commercially available glucuronide derivatives and sulfate were purchased (Calbiochem) and evaluated in the biological assays. Similar intermediates could be used for the synthesis of glucopyranose, mannopyranose and galactopyranose derivatives.

Optical rotations were determined with a Perkin-Elmer 241 model polarimeter at the sodium D line at 23°C. NMR spectra were recorded with

JEOL JNM-GX270 and Varian Inova 300 and 500 spectrometers. Chemical shifts are reported relative to internal tetramethylsilane in chloroform (δ 0.0), deuterium oxide (δ 4.8) or acetonitrile (δ 1.94) for Η and either chloroform (δ 77.0), deuterium oxide (with acetonitrile as reference, δ 119.2) for ¹³C. Coupling constants are reported in hertz. IR spectra were recorded with a

Mattson Galaxy Series FTIR 3000 on KBr discs or for liquid film. Melting points were measured on a Gallenkamp Melting Point apparatus. Elemental analysis was performed on an Exeter Analytical CE440 elemental analyser. High resolution mass spectra were measured on either a VG Micromass 70/70H or VG ZAB-E spectrometer. TLC was performed on aluminium sheets pre-coated with Silica gel 60 (HF₂5 , Merck) and spots visualized by UV and/ or charring with sulphuric acid-ethanol (1:20). Flash Column

Chromatography was carried out with Silica gel 60 (0.040-0.630 mm, Merck) and employed a stepwise solvent polarity gradient correlated with TLC mobility. Preparative HPLC was carried out using a Waters system using a C-4 RP-HPLC column eluting with HPLC grade water and MeCN solvent mixtures. Chromatography solvents used were ethyl acetate (Riedel- deHaen) and petroleum ether (BDH laboratory supplies, fraction of light petroleum ether with boiling point 40-60 °C). Acetonitrile, toluene, benzene and dichloromethane reaction solvents were freshly distilled from calcium hydride.

Intermediate 1: 1,2,3,4-Tetra-O-acetyl-β-D-glucopyτanuronic acid, methyl ester (Von Roedern et al, (1996), J. Am. Chem. Soc. 118: 10156-67). Glucurono-3,6-lactone (43.0 g, 240 mmol) was suspended in dry methanol (700 mL) and dimethylethylamine (0.5 mL) was added. The reaction was stirred for 12 h until all of the lactone had dissolved. The solvent was evaporated and the foam was used without purification. Acetic anhydride (210 mL, 2.2 mol) and sodium acetate (21.0 g 260 mmol) were added and the suspension was stirred for 8 days. The reaction was poured onto 1 L of ice water and stirred overnight. The β-acetate was separated by filtration, washed with water and recrystallised from ethyl acetate/ petroleum ether to give the title compound (43.8 g, 47 %):Η NMR (300 MHz, OMSO-d₆) δ 6.02 (d, 1H, J 8.2, H-l), 5.51 (apt t, 1H, J 9.6, H-3), 5.06-4.95 (overlapping signals, 2H, H-4, H-2), 4.63 (d, 1H, J 9.6, H-5), 3.64 (s, 3H, OCH3), 2.08-1.97 (4s, 12H,

Intermediate 2: l-Bromo-2,3,4-tri-O-acetyl- -D-glucopyranuronic acid, methyl ester (Bollenback et al (1955), J. Am. Chem. Soc. TA. 3310-3314).

Intermediate 1 (2.0 g, 4.52 mmol), was dissolved in 30% HBr-AcOH (8.0 mL) and allowed to stand overnight at 4 °C. The solvent was removed under reduced pressure and the residue was dissolved in chloroform (4.0 mL). The organic layer was washed with cold saturated aqueous sodium carbonate water, dried (Na2S0 ) and the solvent removed. Recrystallisation of the residue from absolute ethanol gave the title compound (0.93 g, 46 %): Η NMR (300 MHz, CDC1₃) δ 6.64 (d, 1H, J 4.1, H-l), 5.62 (apt t, 1H, J 10, H-3), 5.25 (dd, 1H, J 9, 10, H-4), 4.86 (dd, 1H, J 4, 10, H-2), 4.58 (d, 1H, J 9, H-5), 3.76 (s, 3H, OCH3), 2.10-2.05 (3s, each 3H, each CH3CO).

Intermediate 3: 2,3,4-Tri-O-acetyl-D-glucopyranuronic acid, methyl ester

(Tietze and Seele, Carbohydr. Research (1986) 148: 349-352) Water (1.2 mL) and Ag₂C0₃ (1.8 g, 6.5 mmol) were added to a stirred solution of intermediate 2 (2.59 g, 6.5 mmol) in acetone (6.0 mL) at 0 °C. The mixture was stirred for two hours, filtered through celite and the solvent removed under reduced pressure. The residue was then purified by chromatography to give the title compound (1.75 g, 81%): Η NMR (300 MHz, CDCI₃) δ 5.61-5.30 (m, 2H, H-l, H-4), 5.18 (dd, 1H, J 9, 10, H-3), 4.95

(dd, 1H, J 4,10, H-2), 4.59 (d, 1H, J 10, H-5), 3.74 (s, 3H, OCH3), 2.02, 2.03, 2.04

Intermediate 4: l- -(Trichloroacetimidoyl)-2,3,4-tri-O-acetyl-α-D- glucopyranuronic acid, methyl ester (Brown et al, J. Chem. Research (1997), 370-371).

A solution of intermediate 3 (4.54 g, 13.6 mmol) in dichloromethane (68 mL) and trichloroacetonitrile (9.8 mL) was stirred at 20 °C with potassium carbonate (10.3 g) for 16 h. The reaction mixture was purified by column chromatography. The isolated product was recrystallised from ethyl acetate/ hexane to give the title compound 3.3 g (50.7 %); Η NMR (300 MHz, CDCls) δ 8.74 (s, 1H, NH), 6.64 (d, 1H, J 4, H-l), 5.63 (apt t, 1H, J 10.0, H-4), 5.27 (apt t, 1H, J 10.0, H-3), 5.15 (dd, 1H, J 4, 10.0, H-2), 4.50 (d, 1H, J 10, H-5),

3.75 (s, 3H, OCH3), 2.01, 2.04, 2.05 (3s, 9H, CH3CO).

Intermediate 5: l-Azido-2,3,4-tri-O-acetyl-β-D-glucopyranuronic acid, methyl ester (Von Roedern et al (1996), J. Am. Chem. Soc. 118: 10156-670). Trimethylsilyl azide (15.5 mL, 190 mmol) was added to a stirred solution of

Intermediate 1 (31 g, 82 mmol) in dry CH₂C1₂ (450 mL) and SnCl (4.0 mL, 29 mmol). The solution was stirred at room temperature for 3h, then diluted with CH₂C1₂ (300 mL) and washed with 10 % K₂COs (3 x 100 mL) and twice with brine (50 mL). After drying (Na2S0 ), the solution was concentrated in vacuo and recrystallised from EtOAc / petroleum ether to give the title compound (29.6 g, 63.5 %): Η NMR δ (300 MHz, CDCI3) 5.40 (apt, t, 1H, J 9.6, H-3), 5.20 (d, 1H, J 8.8, H-l), 5.06 (apt. t, 1H, J 9.8, H-4), 4.87 (apt. t, 1H, J 9, H-2), 4.57 (d, 1H, J 10.0, H-5), 3.66 (s, 3H, OCH3), 2.04, 1.99, 1.98 (each s, each 3H, OAc). Method A: Procedure for synthesis of O-glycosides

A solution of intermediate 2 (1.0 eq) in dichloromethane was added to a stirred solution of the appropriate alcohol (0.66 eq), silver carbonate (2.25 eq), silver perchlorate (0.1 eq) in dichloromethane over activated 4A molecular sieves, under nitrogen. After 12 h the reaction was diluted with dichloromethane, filtered through celite, washed with water, dried (sodium sulfate anhydr.) and the solvent removed. The residue was purified by chromatography.

Method B: Procedure for synthesis of O-glycosides

The alcohol (1.0 eq) and intermediate 4 (1.0 eq) in anhydrous dichloromethane over activated 4A molecular sieves was placed under a nitrogen atmosphere and cooled to -15 °C. Boron trifluoride, diethyl etherate (0.5 eq in CH2CI2) was added and the mixture stirred for 12 h. The reaction mixture was then diluted with dichloromethane, washed with Na₂C0₃ (aq.) and concentrated and the residue was purified by chromatography.

Method C: Procedure for reaction of glycosyl-azides and acid chlorides promoted by phosphines (Boullanger et al (2000), Carbohydr. Res. 324: 97- 106)

To a mixture of intermediate 5 (1.0 mmol) and the acid chloride (2.0 mmol), dissolved in anhydrous acetonitrile (4 mL), was added dropwise a solution of triphenylphosphine (1.3 mmol) in anhydrous dichloromethane (1 mL) at room temperature. The reaction was allowed to stir for 12h, then diluted with 20 mL CH₂C1₂, washed with a saturated solution of NaHCO₃ (5 mL) and then with water until neutral pH was achieved. After drying over Na₂S0₄, the solution was filtered, evaporated to dryness and then purified by chromatography .

Method D: Preparation of acid chlorides Thionyl chloride (2 eq) was added to the carboxylic acid (1 eq) in dry toluene at 0 °C. The reaction was allowed to come to room temperature, heated at 70 °C for 3h. The acid chlorides were purified by distillation.

Method E: Preparation of acid chlorides A solution (1.5 M) of thionyl chloride (5.46 mL, 0.075 mol) and benzotriazole

(8.93 g, 0.075 mol) was prepared in dry dichloromethane. The reaction was carried out by adding 1.25 mmol equivalent of this solution drop wise to a stirred solution of carboxylic acid (1 mmol) in dry dichloromethane (20 mL). The reaction mixture was stirred for 10 minutes, after which the precipitated benzotriazole hydrochloride salt was filtered off. The filtrate was stirred with

MgS0₄.7H₂0 (~ 0.5 g) to destroy excess reagent. The solids were then filtered off and the acid chloride was recovered by removal of solvent.

Method F: Procedure for synthesis of Intermediate 6 and its coupling reaction with carboxylic acids.

Treatment of intermediate 5 with Pd/C in dry THF at -5°C under H₂ gave intermediate 6 and its α-anomer in a 13:1 ratio. *H NMR δ (300 MHz, CDC1₃) 5.30 (apt t, 1H, J 9.6, H-3), 5.16 (apt t, 1H, J 9.6, H-4), 4.86 (apt t, 1H, J 9.6, H- 2), 4.71 (d, 1H, J 6.7, H-l), 4.23 (d, 1H, J=8.64, H-l), 4.04 (d, 1H, J 10.0, H-5), 3.75 (s, 3H, OCH3), 2.07, 2.03, 2.02 (each s, 3H, OAc). DCC (1 eq) was added dropwise to HOBT (1 eq), DMAP (1 eq) and intermediate 6 stirred in dry THF at 0 °C. After 12 h the precipitated DCU was filtered off and the solvent removed under reduced pressure. The residue was dissolved in CH₂CI₂, washed with NaC0₃, Brine and dried with NaS0₄.The solution was filtered, evaporated to dryness and the residue purified by chromatography to isolate desired product.

Method G: Procedure for the deprotection of O-glucuronic acids and N- glucuronamides

A solution of 0.1N LiOH in MeOH/ water/ THF (2.5/1.0/0.5) (6 eq) was added to a solution of product obtained from method A-C and method F. The resulting solution was allowed to stir until TLC analysis (3:1

EtOA MeOH) indicated the disappearance of starting material. The solution was then diluted with water and neutralised by adding amberlite. THF was then added to homogenise the suspension. The amberlite was removed by filtration and solid Na₂Cθ3 was added. The solvent was removed under reduced pressure and the residue was purified by column chromatography

(EtOAcMeOH) to give the desired product and further purified by preparative HPLC (C-4 reverse phase).

The following selected experimental procedures are typical of that used or synthesis of the compounds.

Example 1 - succinimidoethyl-β-D-glucopyranuronic acid

N-(2-Hydroxyethyl)succinimide (200 mg, 1 mmol) and intermediate 2 (0.6 g, 1.5 mmol) were reacted according to method A to give succinimidoethyl-

2,3,4-tri-O-acetyl-β-D-glucopyranuronic acid, methyl ester (0.18 g, 40 %): m.p. 195-200 °C; [ ]_D -21.8 (c 0.096, CHC1₃); Η NMR δ (300 MHz, CDC1₃)

5.22 (overlapping signals, 2H, H-3, H-4), 4.94 (apt t, 1H, J 7.0, H-2), 4.58 (d, 1H, J 7.0, H-l), 4.03 (m, 2H, OCH(H), H-5), 3.70-3.08 (ms, 6H, OCHs, CH₂N, CH(H)0), 2.07 (s, 4H, CH₂CH₂C=0), 2.01, 2.04, 2.06 (each s, each 3H, each OAc); ¹³C NMR δ (CDCls) 177.1 (s, C=0, methyl ester), 171.1, 170.1, 169.3 (each s, each C=0), lOO.ld, C-l), 72.5, 72.0 , 71.3, 69.3 (each d, C-2-5), 65.5 (t, C-6), 52.9 (q, OCH3), 38.1 (t, CH₂), 28.2 (t, CH₂), 20.7, 20.6, 20.5 (each q, each

OAc); IR (KBr) 2982, 1759, 1705, 1395, 1273, 748 cmX Anal. Calcd for C₁₉H25θι₂N: C, 49.67, H, 5.70, N, 3.04. Found C, 49.59, H, 5.61, N, 3.04. MS Calcd for M+Na 482.1292; Found 482.1292. This intermediate (58.1 mg, 0.012 mmol) was deprotected as described in Method G to give example 1 (20 mg, 69 %): m.p. 205 °C; [α]_D -3.12 (c 1.6, MeOH); Η NMR δ (300 MHz, D₂0) 4.54

(d, 1H, J 8.0, H-l), 4.11-4.01 (m, 1H, H-4), 3.87-3.73 (m, 2H, OCH(H), H-5), 3.60 (m, 2H, CH ), 3.56-3.46 (m, 2H, H-3, OCH), 3.45-3.37 (m, 1H, H-2), 2.53 (s, 4H, CH₂CH₂C=0); ¹³C-NMR δ (D₂0) 184.0, 178.8, (each s, each C=0), 105.0 (d, C-l), 78.7, 78.2, 75.7, 74.5 (each d, C-2-5), 71.3 (t), 42.04 (d), 35.77 (t), 35.05 (t); MS Found 342.0 (M+Na), required 342.1.

Example 18 - (N-(2-thiophenoyl)-y#-D-glucopyranosylamine) uronic acid

2-Thiophenoyl chloride (0.5 g, 3.4 mmol) and intermediate 5 (0.41 g, 1.14 mmol) were added to anhydrous acetonitrile (4 mL), and triphenylphoshine polystyrene (0.5 g, 3.4 mmol, from Novabiochem) was added at room temperature. The reaction was allowed to stir for 12h, then filtered, diluted with 20 mL CH2CI2, washed with a saturated solution of NaHC0₃ (5 mL) and then with water. After drying over Na₂S0 , the solution was filtered, evaporated to dryness and then purified by chromatography, to afford (N-(2- thiophenoyl)-2,3,4-tri-0-acetyl-/^D-glucopyranosylamine)uronic acid, methyl ester (0.26 g, 53%); [α]²⁰ _D -15.7° (c 0.134, CHC1₃); Η NMR (CDCI3, 300MHz) δ 7.88-7.05 (ms, 3H, aromatic), 7.40 (d, 1H, / = 9.0 Hz, NH), 5.54 (overlapping signals, 2H, H-l and H-3), 5.19 (apt t, IH, /₃, = J₄,₅ = 9.5 Hz, H- 4), 5.09 (apt t, IH, J₂,ι = J₂,3 = 9.5 Hz, H-2), 4.27 (d, IH, /₄,₅ 9.5 Hz, H-5), 3.71 (s, 3H, OMe), 2.07, 2.05, 2.04 (3s, each 3H, 3xOAc); "c NMR (CDC1₃, multiplicity in parenthesis as obtained by DEPT) δ 171.3, 169.8, 169.3, 167.2, 161.9 (each s, each C=0)_f 137.4 (s, aromatic C), 131.8, 129.4, 128.0 (3C, each d, aromatic CH), 78.5 (d, C-l), 73.7, 71.8, 70.4, 69.6 (each d, C-2-5), 52.9 (q, OMe), 20.7, 20.5, 20.4 (each q); IR (liquid film): υ 3354 (NH), 2955, 2358, 1750 , 1659, 1541, 1372, 1232, 1037, 734 cm"¹; HRMS-CI (M/Z, positive ion mode): Found 444.0961, required 444.0964 [M+H]⁺. This intermediate (159 mg, 0.4 mmol) was treated with 6 eq of 0.05 M LiOH in MeOH/ water/ THF

(2.5/1.0/0.5) as described in method G and the resulting solution allowed to stir until TLC analysis (3:1 EtOAc:MeOH) indicated the disappearance of starting material. The solution was then diluted with water and neutralised by adding acidic amberlite. THF was then added to homogenise the suspension. The amberlite was removed by filtration and solid Na₂Cθ3 was added. The solvent was removed under reduced pressure and the residue was purified by column chromatography (EtOAcMeOH) to afford the title compound as an off-white solid (90 mg, 83%). Further purification of a portion of this material by C-4 RP-HPLC (95:5 H₂0/MeCN; isocratic) yielded, after lyophilization, example 18 as an amorphous white solid which was then used for biological evaluation; Η-NMR (D₂0, 270 MHz): δ 7.47 (m, 3H, Ar-H), 5.15 (d, IH, Jι,₂ = 8.4 Hz, H-l), 3.82 (d, IH, J₄,₅ = 9.0 Hz, H-5), 3.56 (m, 3H, H-2, H-3 and H-4); ^C-NMR (D₂0, 75 MHz): δ 178.4 and 168.0 (each s, each C=0), 139.1 (s, aromatic C), 135.4, 133.5 and 131.0 (3C, each d, aromatic CH), 82.4, 80.9, 78.9, 74.5 and 74.4 (5C, C-l-5); ESMS (M/Z, negative ion mode): Found 301, required 301 [M-H]-. Example 24: (N-(4-Oxo-4-phenyl-butyryl)-β-D-glucopyranosylamine) uronic acid

Treatment of 3-Benzoylpropionic acid (0.27 g, 1.5 mmol) and intermediate 5 (0.5 g, 1.5 mmol) as described in method F gave (N-(4-oxo-4-phenyl-butyryl)-

2,3,4-tri-0-acetyl-β-D-glucopyranosylamine)urorvic acid, methyl ester (0.2, 35 %): m.p. = 95-97 °C; [ ]_D= +2.6° (c 0.01, CHC1₃). Η NMR δ (300 MHz, CDCI3) 7.96-7.28 (ms, 5H,aromatic-H), 6.81 (d, IH, J 10, NH), 5.38-5.31 (overlapping signals, 2H, H-l, H-3), 5.15 (apt t, IH, J 9.5, H-4), 5.03 (apt t, IH, J 9.5, H-2), 4.17 (d, IH, J 10, H-5), 3.72 (s, 3H, OCH3), 3.47 (m, IH, CH(H)),

3.24-3.15 (m, IH, CH(H)), 2.65-2.53 (m, 2H, CH₂); ¹³C NMR (300 MHz, CDCI3) 198.0, 172.8, 171.3, 169.9, 169.7, 167.5 (each s, each C=0), 136.7 (s, aromatic C), 133.5, 128.8, 128.3, 128.3 (each d, each aromatic CH), 78.3, (d, C-l), 74.2, 72.3, 70.4, 69.9 (each d, C-2-5), 53.1 (q, OCH3), 33.6, 30.4 (each t, CH₂), 20.9, 20.8, 20.6 (each q, OAc); IR (KBr) 3355, 2932, 2366, 1753, 1681, 1538, 1449, 1372,

1228, 1099, 1037, 893, 667, 522 cmX The reaction of this compound (100 mg, 0.23 mmol) as described in method G gave example 24. Example 24 was purified by HPLC (C-4 RP, flow rate 10.0 ml/s, 95:5, water:acetonitile) before biological evaluation. [ ]_D= -18.0° (c 0.05). Η NMR δ (300 MHz, D₂0) 5.06 (d, IH, J 9.0, H-l), 3.89 (d, J 9.0, H-5), 3.68 (ms, 6H, H-2, H-3, H-4, CH₂), 2.81

(t, 2H, J 6.7); "C NMR (300 MHz, D₂0) 203.5, 176.7, 175.9 (each s, each C=0), 136.3 (s, aromatic C), 134.3, 129.6, 129.1, 128.4, 128.4 (each d, each aromatic CH), 79.4 (d, C-l), 78.0, 76.5, 72.0, 72.0 (each d, C-2-5), 33.8, 30.0 (each t, CH₂); IR (KBr) 3441, 2910, 2343, 1647, 1448, 1211, 1038, 595 cπrl; LRMS [M-H]- ³⁵2- Example 32 N,N'-Di-( ^D-galactopyranosyl)-terephthalamide

2,3,4,6-Tetra-0-acetyl- D-galactopyranosylarnine was first prepared. β-D- Galactose pentaacetate (13.0 g, 33 mmol) was suspended in dry CH₂C1₂ (30 mL). TMS-Ns (4.42 mL, 33 mmol) and SnCl (1.93 mL, 16.5 mmol) were added and the reaction mixture was allowed to stir at rt. TLC analysis (EtOAc: petroleum ether, 1:1) showed the reaction was complete after 1 h. The reaction mixture was washed with sodium bicarbonate (3 x 50 mL), water (3 x 50 mL) and dried (MgS0₄). Excess solvent was removed to give a white solid (12.3 g, 100%). This azide (15.71 g, 42 mmol) was suspended in ethanol (200 mL) and Pd-C (1.18 g) was added. The reaction vessel was shaken under a hydrogen atmosphere at a pressure of 3 atm. Analysis by TLC (EtOAc: petroleum ether, 1:1) showed that the reaction was complete after 24 h. The catalyst was filtered off and excess solvent was removed and the residue was purified by chromatography (EtOAc: petroleum ether,

1:1) to give the amine as a white solid (9.96 g, 68%); [ ]_D +29.5° (c 1.0, MeOH); mp 134-136 °C; Η-NMR: δ (300 MHz, CDCls) 5.40 (dd, IH, J ,₅ 1.2, /₄,3 3.06, H-4), 5.09-5.00 (m, 2H, H-2, H-3), 4.16 (d, IH, Jι,₂ 8.2, H-l), 4.11 (overlapping signals, 2H, H-6a, H-6b), 3.90 (apt dt, IH, /s, 1.2, /5,6a = /5,6b = 6.5, H-5). Terephthalic acid (0.17 g, 1.0 mmol), EDC (0.38 g, 2.0 mmol) and

DMAP (catalytic) were suspended in dry dichloromethane (10 mL). The reaction mixture was stirred at rt. for 30 minutes, and then the amine (0.7 g, 2.0 mmol) was added. The reaction was allowed to stir at rt. TLC analysis (EtOAc) showed the reaction was complete after 72 h. Excess solvent was removed and the residue purified by chromatography (EtOAc: petroleum ether, 3:1) to yield N,N'-Di-(tetra-0-acetyl- ?-D-galactopyranosyl)- terephthalamide (0.1 g, 0.12 mmol), as a white foam (0.31 g, 19%, mixture of anomers); R/0.54 (EtOAc: petroleum ether, 3:1); [α]_D +50.0° (c 0.02, CHCls); Η-NMR: δ (300 MHz, CDCI3, 1:4 mixture of aβββ anomers) 7.85 (m, 4H, aromatic H), 7.49 (d, IH, /NH,H-I 8.0, NH, aβ< om.eτ), 7.23 (d, IH, JN_H,_HI 9.0, NH, yø 2-anomer), 6.17 (br signal, IH, H-l, α^anomer), 5.20-5.54

(overlapping signals, H-l, ββεnxomer, H-2— 4), 4.09-4.17 (overlapping signals, H-5, H-6a, H-6b), 2.04-2.20 (overlapping signals, OAc); ¹³C-NMR: δ (CDCI3) 172.1, 170.6, 170.2, 170.0 (each s, each CO, ?/?-anomer), 171.4, 171.0, 170.8, 170.4 (each s, each C=0, c^-anomer), 166.5 (s, aromatic C=0, aβanomer), 166.3 (s, aromatic C=0, ββaixomeτ), 137.3 (s, aromatic C, αβ anomer), 136.5 (s, aromatic C, ?/?-anomer), 128.1, 127.9 (2 signals), 127.8 (each d, aromatic C), 79.5, 72.7, 70.9, 68.9, 67.5 (each d, /^anomer), 69.0, 67.8, 67.7, 66.6 (each d, α -anomer), 61.8 (t, αr/J-anomer), 61.4 (t, /?/?-anomer), 21.0, 20.9, 20.8 (2 signals) (each q, each OAc); v_max (KBr) 2972, 2933, 2356, 2336, 1752, 1673, 1547, 1501, 1368, 1229 cmX HRMS-FAB: found 847.2385

[M+Na]⁺, required 847.2398. The isomerisation of the β-amine, which leads to formation of /β mixture can be precluded by use of freshly prepared amine.

This product was suspended in MeOH (10 mL). Sodium methoxide (0.1 mL of a 0.25 M solution) was added and the reaction mixture was allowed to stir at rt. TLC analysis (MeOH) after 3h showed that the reaction was gone to completion. Amberlite (H⁺) was added and after 5 min the reaction mixture was filtered and the solvent removed. The residue was purified by chromatography (MeOH: EtOAc, 1:1) to yield example 32 as a white solid (0.04 g, 67%, mixture of anomers); R/ 0.21 (MeOH); [α]_D +65.0° (c 0.04, H₂0); mp 60-64°C; Η-NMR: δ (300 MHz, D₂0, 1:3 mixture of aβ.ββ anomers) 8.02

(s, 4H, aromatic H), 5.92 (d, IH, Jι,₂ 5.7, H-l, o^anomer), 5.24 (d, IH, Jι ₂ 8.9, H-l, /?/?-anomer), 4.25 (dd, IH, J₂,ι 5.7, J₂₃ 10.3, H-2, α/?-anomer), 3.76-4.11 (overlapping signals, 11H, H-2, /?/?-anomer, H-3, H-4, H-5, H-6a, H-6b, aβ and ^anomers); ¹³C-NMR: δ (D₂0) 174.9 (s, C=0, αβ-anomer), 173.9 (s, C=0,

139.9 (s, aromatic C, α/?-anomer), 139.4 (s, aromatic C, ββ anomer), 130.8 (d, aromatic C, α^-anomer), 130.7 (d, aromatic C, ββdxnomeτ), 83.2 (d, C-l, yflyS-anomer), 80.4 (d, C-l, αβ-anomer), 79.8, 76.3, 72.2, 71.6 (each d, ββεLixomeτ), 75.0, 72.2, 71.7, 69.3 (each d, aβεmomeτ), 64.0 (t, α/?-anomer) 63.8 (t, ^anomer); i (KBr) 3410, 2931, 1660, 1550, 1424, 1299, 1086 cm-¹; FABMS 511 [M+Na]⁺.

Example 30: Thiophene-2-carboxylic acid-N-( ?-D-glucopyranosyl)-amide

2,3,4,6-Tetra-0-acetyl- ^D-glucopyranosylamine was prepared first of all. 2,3,4,6-Tetra-0-acetyl-y#-D-glucopyranosyl bromide (38.8 g, 94.0 mmol), sodium azide (24.5 g, 380 mmol) and tetrabutylammonium hydrogen sulphate (31.9 g, 94.0 mmol) were suspended in a two-phase solution of CH₂Cl₂/NaHC0₃ (100 mL, 1:1). The reaction mixture was stirred at rt. TLC analysis (EtOAc: petroleum ether, 1:1) showed that the reaction was complete after 3 h. The organic layer was washed with water (3 x 100 mL) and sodium bicarbonate (3 x 100 mL), dried (MgS0 ), excess solvent was removed and the residue was purified by chromatography (EtOAc: petroleum ether, 1:4) to yield the β-azide as a white solid (36.9 g, 74%); R/ 0.64 (acetone: CH₂C1₂, 1:4); Η-NMR: δ (300 MHz, CDC1₃) 5.22 (apt t, IH, /₃,₂

= /₃, = 9.5, H-3), 5.10 (t, IH, /₄,₃ = /4,s = 9.5, H-4), 4.96 (apt t, IH, J₂,ι = J₂,₃ = 9.5, H-2), 4.65 (d, IH, /ι,₂ 9.5, H-l), 4.28 (dd, /_6a,6b 12.4, /_6a,₅ 4.8, H-6a), 4.17 (dd, /₆b,6a 12.4, J₆ ,5 2.4, H-6b), 3.80 (ddd, J₅₆a 4.8, /5,6b 2.4, J₅ 9.5, H-5), 2.10, 2.08, 2.03, 2.01 (each s, each 3H, each OAc). This azide (5.0 g, 13.4 mmol) was suspended in ethanol (50 mL) and Pd-C (2 spatulas) was added. The reaction vessel was shaken under a hydrogen atmosphere at a pressure of 3 atm. Analysis by TLC (EtOAc) showed that the reaction was complete after 24 h. The catalyst was filtered off and excess solvent was removed. The residue was purified by chromatography (EtOAc: petroleum ether, 1:1) to give the amine as a white solid (3.12 g, 67%); R/ 0.42 (EtOAc); Η-NMR: δ (300

MHz, CDCI3) 5.24 (apt t, IH, /₃₂ = J₃₄ = 9.6, H-3), 5.03 (apt t, IH, / ₃ = J _/5 = 9.6, H-4), 4.82 (dd, IH, /₂,ι 8.9, /₂,3 9.6, H-2), 4.22 (dd, 2H, /_6a,6b 12.7, /_6a,5 4.4, H- 6a), 4.18 (brs, IH, H-l), 4.11(dd, IH, J_6a,6b 12.4, /₆b,5 2.4, H-6b), 3.69 (m, IH, H- 5), 2.10 (2 signals), 2.02, 2.01 (each s, each 3H, each OAc). Thiophene-2-carboxylic acid (0.07 g, 0.58 mmol), DCC (0.14 g, 0.7 mmol) and DMAP (catalytic) were suspended in dry dichloromethane (20 mL) and the reaction mixture was allowed to stir at rt for lh. 2,3,4,6-Tetra-O- acetyl-/^D-glucopyranosylamine (0.2 g, 0.58 mmol), was then added and the reaction mixture was allowed to stir at rt. TLC analysis (EtOAc) showed that the reaction was complete after 24h. The solvent was removed and the residue purified by chromatography (EtOAc: petroleum ether, 1:1) to yield thiophene-2-carboxylic acid-N-(2,3,4,6-tetra-0-acetyl- ?-D- glucopyranosyl)-amide as a white solid (0.1 g, 41%); R/ 0.22 (EtOAc); [c +15.0° (c 0.02 CHCI3); Η-NMR: δ (300 MHz, CDCI3) 7.55 (dd, IH, J 1.1, J 5.0, thiophene H), 7.51 (dd, IH, / 1.1, / 3.7, thiophene H), 7.09 (dd, IH, / 3.8, / 4.9, thiophene H), 7.01 (d, IH, /NH,H-I 9.0, NH), 5.34 (apt t, IH, / = /_H-_I,_NH = 9.0, H-l), 5.38 (apt t, /₃₂ = /₃,₄ = 9.4, H-3), 5.08 (2 x overlapping apt t, 2H,

H-2, H-4), 4.34 (dd, IH, /_6a,6b 12.5, J_6a,5 4.4 , H-6a), 4.11 (dd, IH, J₆b,6_a 12.7, /₆b,5 2.3 , H-6b), 3.90 (ddd, IH, /₅₆a 4.4, /5,6b 2.3, /₅,₄ 10.0, H-5), 2.08, 2.05, 2.04 (2 signals) (each s, each 3H, each OAc) ; ¹³C-NMR: δ (CDCI3) 171.6, 170.6, 169.8, 169.6 (each s, each C=0), 161.7 (s, thiophene C=0), 137.5 (s, thiophene C), 131.7, 129.2, 127.9 (each d, thiophene C), 79.0, 73.7, 72.6, 70.8,

68.3 (each d), 61.7 (t), 20.7 (3 signals), 20.6 (each q, each OAc); Umax (KBr) 3331, 2928, 2851, 1753, 1626, 1753, 1626, 1576, 1536, 1435, 1368, 1244 cmø HRMS-CI: Found 458.1121 [M+H]⁺, required 458.1119. This intermediate (0.06 g, 0.13 mmol) was suspended in MeOH (5 mL). NaOMe (0.1 mL of a 0.25 M solution) was added. The reaction was not gone to completion after

1.5h so another 0.1 mL of NaOMe was added. Analysis by TLC (MeOH: EtOAc, 1:4) showed that the reaction was complete after 2h. The reaction mixture was filtered and the solvent removed to yield example 30 as a clear oil (0.02 g, 50%); R/ 0.23 (MeOH: EtOAc, 1:4); Η-NMR: δ (300 MHz, D₂0, 1:7 mixture of cc.β anomers) 7.89 (dd, IH, / 1.1, / 3.9, thiophene H), 7.86 (dd,

IH, / 1.2, / 5.1, thiophene H), 7.29 (dd, IH, / 3.8, / 4.9, thiophene H), 5.24 (d, IH, Jι ₂ 8.6, H-l, βεxnomeτ), 3.97 (dd, IH, /_6a,5 2.27, /_6a,₆b 12.4, H-6a), 3.83 (dd, IH, /βb,5 5.0, /₆b,6_a 12.4, H-6b), 3.52-3.71 (overlapping signals,'4H, H-2, H-3, H-4, H-5): additional signals for α-anomer δ_H 7.93 (dd, IH, / 1.2, 3.8, thiophene H), 5.84 (d, IH, /_{1 2}4.8, H-l, -anomer); ¹³C-NMR: δ (D₂0) 168.2

(s, C=0), 139.2 (s, thiophene C), 135.6, 133.7, 131.2 (each d, each thiophene C), 82.8, 80.5, 79.4, 74.5, 72.1 (each d), 63.4 (t); additional signals for a- anomer δc 135.4, 133.8, 131.1 (each d, each thiophene C), 78.6, 75.8, 75.6, 72.4, 72.2 (each d). The product was further purified by RP-HPLC (C-4) to give only the β-anomer which was used for biological evaluation.

Example 33 N-(y9-D-mannopyranosyl)benzamide

2,3,4,6-Tetra-0-acetyl- ^D-mannopyranosylamine was prepared first of all. Penta-O-acetylmannopyranose (45.14 g, 0.12 mmol) was suspended in dry dicholoromethane (100 mL). TMS-N₃ (15.4 mL, 0.12 mmol) and SnCl (7.02 mL, 0.06 mmol) were added and the reaction was allowed to stir at rt. under an inert atmosphere. TLC analysis (EtOAc: petroleum ether, 1:1) showed that the reaction was complete after 24 h. The reaction mixture was washed with sodium hydrogen carbonate (3 x 50 mL) and water (3 x 50 mL), dried (MgS0₄) and excess solvent removed. The residue was purified by chromatography (EtOAc: petroleum ether, 1:5) to yield the α- azide as a yellow syrup (31.2 g, 70%); R/0.34 (EtOAc: petroleum ether, 1:1); Η-NMR: δ (300 MHz, CDC1₃) 5.31 (br s, IH, H-l), 5.04-5.25 (overlapping signals, 3H, H-2-4), 4.21 (dd, IH, J_6a,6b 12.3, J_{6a 5} 5.3, H-6a), 4.07 (overlapping signals, 2H, H-5, H-6b), 2.07, 2.02, 1.96, 1.90 (each s, each 3H, each OAc); "C-NMR: δ (CDC1₃) 170.6, 170.0, 169.8, 169.7 (each s, each C=0),

87.6 (d, C-l), 70.8, 69.3, 68.4, 65.8 (each d), 62.3 (t), 20.9, 20.8, 20.7 (2 signals) (each q, each OAc); υ_max (film) 3009, 2944, 2891, 2124, 1750, 1646, 1445, 1378, 1249 cm"¹. This azide (8.0 g, 21.4 mmol) was suspended in EtOAc (10 mL) with some EtOH (5 mL) for dissolution. Raney nickel (3 spatulas) was added and the reaction mixture shaken under an atmosphere of hydrogen at a pressure of 50 psi. TLC analysis (EtOAc) showed the reaction was complete after 24 h. The reaction mixture was filtered carefully (care was taken to prevent Raney nickel going dry as this is a fire hazard) and the solvent removed. The residue was purified by recrystallisation from 2- propanol to yield the β-amine as a white solid (3.5 g, 47%); R/0.21 (EtOAc); [o]_D -11.6° (c 0.5, CHCls); m.p. 150-154 °C; -NMR: δ (300 MHz, CDC1₃) 5.40 (dd, IH, J₂,ι 1.3, 3.4, H-2), 5.18 (apt t, H, J₄₃ = /₄,5 = 10.0, H-4), 5.07 (dd, IH, J₃,₂ 3.4, /₃₄ 10.1, H-3), 4.46 (br s, IH, H-l), 4.25 (dd, IH, J_6a,₅ 5.6, J_6a,6b 12.3, H-6a), 4.11 (dd, IH, J₆b,5 2.4, J_6b,_6a 12.3, H-6b), 3.67 (ddd, IH, J_{5 6}b 2.4,

JS 5.6, JSΛ 10.0, H-5), 2.20, 2.11, 2.05, 1.98 (each s, each 3H, each OAc); *³C- NMR: δ (CDC ) 170.9, 170.4, 170.2, 169.9 (each s, each C=0), 82.4 (d, C-l), 73.5, 72.2, 70.9, 66.1 (each d), 63.1 (t), 21.0, 20.9 (2 signals), 20.8 (each q, each OAc); Cι H₂₁N0₉: C, 48.40; H, 6.09; N, 4.03. Found C, 48.76; H, 6.08; N, 3.93. Benzoic acid (0.17 g, 1.44 mmol), DCC (1.73 mL, 1.73 mmol of a 1.0 M solution in CH₂C1₂) and DMAP (catalytic) were suspended in dry dichloromethane (20 mL) and the reaction mixture was allowed to stir at rt for 30 minutes. The β-amine (0.5 g, 1.44 mmol) was then added and the reaction mixture was allowed to stir at rt. The reaction was not complete after 24 h so another 1.2 eq of DCC was added. TLC analysis (EtOAc) after a total of 48 h showed that the reaction was complete. The solvent was removed and the residue purified by chromatography (EtOAc: petroleum ether, 1:1) to yield N-(2,3,4,6-tetra-0-acetyl-β-D-mannopyranosyl)- benzamide as a white solid (0.3 g, 45%); R/ 0.53 (EtOAc); [α]_D +1.67° (c 0.2, CHCI3); mp 50-54 °C; -NMR: δ (300 MHz, CDCb) 7.72 (d, 2H, / 8.2, aromatic H), 7.38-7.55 (m, 3H, aromatic H), 7.07 (d, IH, / H,H-I 9.0, NH), 5.78 (d, IH, /_{1 2}, 9.0, H-l), 5.19-5.47 (overlapping signals, H-2-4), 4.32 (dd, IH, /όa,5 4.9, J_6a6b 12.4, H-6a), 4.10 (dd, 2H, /_6b,6a 7.3, /_6b,5 1.1 H-6b), 3.87 (m, IH, H-5), 2.07 (2 signals), 2.06, 2.05 (each s, each 3H, each OAc); ¹³C-NMR: δ (CDCI3) 170.9, 170.8, 170.0, 169.9, 166.9 (each s, each C=0), 133.3 (s, aromatic C), 132.5, 128.9, 127.5 (each d, each aromatic C), 76.9, 74.5, 71.8, 70.7, 65.6 (each d), 62.5 (t), 21.1, 20.9 (2 signals), 20.7 (each q, each OAc); υ_maχ (KBr) 3331, 2966, 2935, 2258, 1751, 1667, 1528, 1434, 1370, 1104, 1054 cm-0 HRMS-CI: found 452.1557 [M+H]⁺, required 452.1559. This compound (0.14 g, 0.31 mmol) was suspended in MeOH (20 mL). NaOMe (0.1 mL of a 0.25 M solution) was added. TLC analysis (MeOH: EtOAc, 1:4) showed that the reaction was complete after 50 minutes. Amberlite (H⁺) was added and after 5 minutes the reaction mixture was filtered and the solvent removed. The residue was purified by chromatography (MeOH: EtOAc, 1:4) to yield the title compoimd as a white solid (0.02 g, 24%); R/ 0.14 (MeOH: EtOAc, 1:4); [α]_D +192.5° (c 0.04, H₂0); Η-NMR: δ (300 MHz, D₂0) 7.87 (d, 2H, / 7.2, aromatic H), 7.73 (t, IH, / 7.4, aromatic H), 7.62 (t,

2H, / 7.6, aromatic H), 5.73 (d, IH, Jι ₂ 2.1, H-l, βaixomer), 3.72-4.19 (overlapping signals, 6H, H-2-6) ¹³C-NMR: δ (D₂0) 172.4 (s, C=0), 133.5 (s, aromatic C, y#-anomer), 133.1 (s, aromatic C, α-anomer) 132.8, 129.0, 127.8 (each d, aromatic C, ?-anomer), 128.4 (d, aromatic C, α-anomer), 79.6, 74.6, 70.8, 69.8, 67.1 (each d, /?-anomer), 78.6, 70.5, 66.7 (each d, α-anomer), 61.2

(t, α-anomer), 61.0 (t, ^anomer); i (KBr) 3400, 2526, 1665, 1558, 1109, 1098 cm-¹. HRMS-FAB: found 306.0954 [M+Na]⁺, required 306.0950. This compound was further purified by semi-prep HPLC (C-4 column; 5:95 AcCN: H₂0) before biological evaluation.

Examples 1-4 were prepared by Method A followed by Method G Example 4 was also prepared by Method B followed by Method G. Examples 5-11, 13, 14, 15, 17, 20 were prepared by Methods C and G. Examples 11,12,16,18, 19, 21-28, 30-33 were most efficiently prepared by Methods F and G. Example 29 was prepared as described for Example 30 using benzoic acid instead of thiophene carboxylic acid.

Analytical and spectroscopic data

Example 2: Phthalimidoethyl-β-D-glucopyranuronic acid.

M.p. 59 °C; [ ]_D -20.8° (c 0.024, MeOH); Η NMR δ (300 MHz, D₂0) 7.66-7.49 (m, 4H, aromatic), 4.57 (d, IH, J 8.0, H-l), 4.25-4.05 (m, IH, OCH(H)), 4.00- 3.81 (m, IH, OCH(H)), 3.72-3.40 (ms, 5H, H-35, CH₂), 3.21-3.32 (apt. t, J 8.0, H-2); ³C-NMR δ (300 MHz, D₂0) 178.7, 178.5, 175.9 (each s, each C=0), 140.2,

137.0 (each s, each aromatic C), 133.1, 132.2, 130.6, 129.9 (each d, each aromatic CH), 105.1 (d, C-l), 78.7, 78.1, 75.6, 74.5 (each d, C-2-5), 71.2 (t); i (KBr) 3417 (OH), 1570, 1417,1056, 661cm-¹; LRMS 390 [M+Na]⁺.

Example 3 [l,4-Dioxaspirol[4,5]dec-2-yl]-β-D-glucopyranuronic acid.

M.p. 75-80 °C; [α]_D -22.7° (c 0.30, MeOH); Η NMR δ (300 MHz, D₂0) 4.56- 4.53 (m, 2H, H-l, CH), 4.51- 3.37 (ms, 7H, CH₂, CH₂, H-3-5), 3.46-3.36 (m, IH, H-2), 1.80 (m, 10H, 5 x CH₂); ¹³C NMR δ (300 MHz, D₂0) 178.6 (s, COOH), 114.0, 113.9 (each s), 105.1, 105.0 (each d, C-l), 78.8, 78.1, 77.0, 76.6, 75.7, 75.6, 74.5 (each d, C-2-5), 73.4, 72.8, 67.7, 38.1, 38.0, 36.4, 27.2 (each t); υ_max (KBr) 3424, 2928, 1617, 1420, 1284, 1054, 663cm-¹; LRMS Found 347.0 [M-H]-, required 347.14.

Example 4: 3-Benzoylphenyl-β-D-glucopyranuronic acid

M.p. 60 °C; [O]Ό -4.2 (c 0.26, MeOH); Η NMR δ (300 MHz, D₂0) 7.68-7.38 (ms, 9H, aromatic H), 5.05 (br s, IH, H-l), 3.79 (br s, H, H-5), 3.69 (ms, 3H,

H-2-5), ³C NMR δ (300 MHz, D₂0) 202.6 (s, C=0), δ 178.0 (s, COOH), 159.0, 140.8, 139.1 (each s), 136.4, 133.0, 132.8, 131.3, 127.7, 124.5, 120.8 (each d, each aromatic CH), 102.5 (d, C-l), 78.9, 78.1, 75.4, 74.5 (each d, C-2-5); υ_max (KBr) 3400, 2907, 1625, 1435, 1204, 1062cm-¹; LRMS Found 373.0 [M-H]-, required 373.1.

Example 5: (N-(4-Chlorophenylacetyl)^β-D-glucopyranosylamine)uronic acid

M.p. 81 °C; [ ]_D -19.6° (c 0.12, MeOH); Η NMR δ (300 MHz, D₂0) 7.70-7.54

(m, 4 aromatic H), 5.18 (d, IH, J 9.0, H-l), 3.99 (d, IH, J 9.0, H-5), 3.91 (br s, 2H, CH₂), 3.78 (m, 2H, H-3,4), 3.68 (apt. t, IH, J 9.0, H-2); C NMR δ (D 0) 175.8 (s, COOH), 174.7 (s, CONH), 134.0, 132.7 (each s, aromatic C), 131.5, 129.01 (each d, each aromatic CH), 79.7 (d, C-l), 77.9, 76.7, 72.1, 72.06 (each d, C-2-5), 41.91 (t, CH₂C=0); υmax (KBr) 3141, 1650, 1567, 1494, 1418, 1302, 736cm-¹; LRMS 344.0 [M-H]-.

Example 6 (N-(3-methyl-2-but-2-enoly)-β-D-glucopyranosylamine)xuOnic acid

M.p. 91-92 °C; [α]_D -45.6 (c 1.1, MeOH); Η NMR δ (300 MHz, D₂0) 5.82 (s, IH, C=CH), 5.05 (d, IH, J 9.0, IH, H-l), 3.68-3.42 (ms, 3H, H-2 -4), 1.96 (s, 6H,

C(CH3)₂); ¹³C NMR δ (D₂0) 178.5 (s, COOH), 173.4 (s, CONH) 158.6 (C=CH), 119.4 (d, C=CH), 81.8 (d, C-l), 80.6, 79.0, 74.4, 74.4 (each d, C-2-4), 29.2, 22.4 (each q, C(CH3)₂); x (KBr) 3423, 2922, 1627, 1419, 1266, 1683, 945, 655cm-¹; LRMS 274.0 [M-H]-.

Example 7: (N-(3-Furan-2-ylacryloyl)-β-D-glucopyranosylamine)uronic acid.

M.p. 170 °C (decomp.); [α]_D-7.5° (c 6.9, MeOH); NMR (300 MHz, D₂0) 7.88 (br s, IH, furan H), 7.68 (d, IH, J 15.0, CH=CH), 7.25 (d, IH, J 3.0, furan H),

6.83 (apt. t, IH, J 2.0, furan H), 6.70 (d, IH, J 15.0, CH=CH), 5.28 (d, IH, J 9.0, H-l), 4.01 (d, IH, J 9.0, H-5), 3.86-3.65 (ms, H-2-4); C-NMR δ (300 MHz, D₂0) 178.5 (s, COOH), 172.31 (s, CONH), 153.3 (s, 0-C=C), 148.2 (d, O- CH=C), 132.7, ,119.1, 118.5, 115.4 (each d, alkene and furan CH), 82.1 (d, C-l), 80.7, 79.0, 74.6, 74.5, (each d, C-2-5); υ_maχ (KBr) 3422, 2925, 1618, 1566, 1419, 1283, 1073, 883, 749cm-¹; LRMS 312.0 [M-H]-.

Example 8: (N-(2-Methylpentanoyl)-β-D-glucopyranosylamine)uronic acid

[ ]_D= -16.6° (c 0.04, MeOH);Η NMR δ (270 MHz, D₂0) 4.79 (d, IH, J=9.2, H- 1), 3.62 (d, IH, J=9.5, H-5), 3.42-3.19 (overlapping signals, 3H, H-2-4), 2.28- 2.03 (ms, 5H, CH, CH₂, CH₂), 0.94 (d, 3H, J=7.0, CHs), 0.70 (t, 3H, J=7.15,

CH₃); ¹³C NMR (300 MHz, D₂0) 181.7, 175.5 (each s, each C=0), 79.0, 78.9 (d, C-l), 77.6, 76.2, 76.2, 71.6, 71.5 (each d, C-2-5), 40.5, 40.4 (each d, CH), 35.5, 35.4 (each t, each CH₂), 16.8, 16.7 (each t, CH₂), 13.1, 13.1 (each q, CHs); υ_max (KBr) 3605, 1555, 1418, 1077, 1021, 639 cm-¹; ES-MS 290 [M-H]-.

Example 9: ((N-cyclopropanecarbonyl)-β-D-glucopyranosylamine)uronic acid.

[α]ϋ= -20.4° (c 0.054, MeOH); Η NMR δ (270 MHz, D₂0) 4.82-4.79 (d, IH, J 9.0, H-l), 3.60 (d, IH, J 9.5, H-5), 3.32-3.24 (overlapping signals, 3H, H-2-4),

1.65-1.47 (m, IH, cyclopropane-H), 0.74 (d, 4H, cyclopropyl-H); ¹³C NMR

(300 MHz, D₂0) 181.2, 178.5 (each s, each C=0), 79.0 (d, C-l), 77.6, 76.1, 71.6, 71.6 (each d, C-2-5), 17.0 (t, CH₂), 10.6, 10.3 (each q, each CH₃); x (KBr) 3374, 569, 1415, 1069, 946, 649cm-¹; UR S 260 [M-H]-.

Example 10: (N-((2,4-Dichlorophenoxy)acetyI)-β-D- glucopyranosylamine)uronic acid.

M.p. 38-43 °C; [α]_D= -7.27° (c 0.055, MeOH) NMR δ (270 MHz, D2O) 7.38 (s, IH, aromatic-H), 7.15 (d, IH, J 9.0, aromatic-H), 6.86 (d, IH, J 9.0, aromatic-H), 4.91 (d, IH, J 9.2, H-l), 3.65 (d, IH, J 9.5, H-5), 3.41-3.32 (overlapping signals, 3H, H-2-4); ¹³C NMR (300 MHz, D₂0) 181.9, 175.4 (each s, each C=0), 151.7 (s, aromatic C), 129.8, 128.0 (each d, each aromatic CH), 126.7, 123.1 (each s, each aromatic C), 115.5 ( d, aromatic CH), 78.7 (d, C-l), 77.8, 76.1, 71.6, 71.5 (each d, C-2-5), 67.8 (t, CH₂); υ_maχ (KBr) 3452 (OH), 2386, 2569, 1417, 1238, 1078, 1019, 604 cm-¹; HRMS Calcd M-l 394.0096; Found 394.0102.

Example 11: (N-(Benzoyl)-y0-D-glucopyranosylamine)iu"onic acid

[α]²⁰ _D +2.5 (c 0.62, D₂0); NMR (D₂0, 270MHz) δ 8.8-7.20 (ms, 5H, aromatics), 5.18 (d, IH, Jι,₂ 8.2, H-l), 4.01 (d, IH, J₄₅ 9, H-5), 3.50 (br s, 3H, H-

2, 3, 4); ¹³C NMR (D₂0, 300MHz) δ 176.0 (s, C=0), 174.6 (s, C=0), 149.9 (s, aromatic), 135.6, 135.4, 131.6, 130.3, 130.2 (5C, each d, each aromatic), 82.6, 79.6, 78.9, 74.2, 74.1 (5C, each d, C-1-5); v_max (KBr) 3399 (OH), 2900, 1649 (C=0), 1644 (C=0), 1258 cm-¹; LRMS 594 [2M]-.

Example 12: (N-(3-Trifluoromethylbenzoyl)-/^D- glucopyranosylamine)uronic acid

[α]_D= -12.3° (c 0.39, H₂0); NMR (D₂0, 270MHz) δ 8.13-7.55 (ms, 4H, aromatics), 5.19 (d, IH, Jι ₂8.4, H-l), 3.83 (d, IH, J₄,s 9.2, H-5), 3.53 (ms, 3H, H- 2, 3, 4); ¹³C NMR (D₂0, 300MHz) δ 173.0 (s, C=0), 170.5 (s, C=0), 133.8 (s, aromatic), 131.4, 129.9, 129.5, 129.5, 124.8, 124.8 (6C, 4 x aromatic C(d), 1 x aromatic C(s) and CF₃), 80.1, 76.6, 76.4, 71.7, 71.5 (5C, each d, C-1-5); υ_ma_x (KBr) 3403, 2903, 1723, 1661, 1551, 1330, 1128, 1074 cm-¹; LRMS: 364 [M - H]-.

Example 13: (N-(3_r5-Dimethylbenzoyl)-y5-D-glucopyranosylamine)tu:onic acid

[α]_D= -10.5° (c 0.2, H2O); NMR (D2O, 270MHz) δ 7.41-7.21 (ms, 3H, aromatics), 5.13 (d, IH, J_w 8.6, H-l), 3.81 (d, IH, J₄,s 9.1, H-5), 3.52 (m, 3H, H- 2, 3, 4), 1.86 (s, 6H, 2xMe); υ_max (KBr) 3431 (OH), 2940, 1723, 1661, 1535, 1312, 1245 cm-¹; LRMS Found 324 [M - H]-. Example 14: (N-(3,4,5-Trimethoxybenzoyl)- M glucopyranosylamine)uronic acid

[O Ό= -48.81° (c 0.29, H₂0); NMR (D₂0, 270MHz) δ 7.03 (s, 2H, aromatics), 5.17 (d, IH, Jι ₂ 8.6, H-l), 3.80 (d, IH, J ,₅ 10.4, H-5), 3.56 (m, 3H, H-2-4), 3.29 (s, 9H, 3xOMe); ¹³C NMR (D₂0, 300MHz) δ 175.9 (C=0), 170.6 (C=0), 140.5, 129.1 (3C, each s, aromatic), 106.9 (2C, each d, aromatic), 80.1, 78.2, 76.6, 72.1, 71.9 (C-l-C-5), 61.2, 56.5 (3C, each q, OCH3); max (KBr) 3409, 1524, 1585, 1416, 1238, 1128, 1077 cm-¹; LRMS 386 [M - H]-.

Example 15: (N-(Biphenyl-2-carbonyl)-5-D-glucopyranosylamine)uronic acid

[ct]_D= -25.1° (c 0.6, H 0); NMR (D₂0, 270MHz) δ 7.60-7.35 (ms, 9H, aromatics), 4.96 (d, IH, Jι,₂ 9.2, H-l), 3.73 (d, IH, J₄,₅ 9.2, H-5), 3.48 (m, 3H, H-

2, 3, 4); υmax (KBr) 3407, 2346, 1881, 1652, 1572, 1420, 1075, 774 cm-¹; LRMS 372 [M - H]-.

Example 16: (N-(2-Phenylquinoline-4-carbonyl)- ?-D- glucopyranosylamine)uronic acid

[α]_D= -8.6° (c 0.11, MeOH); NMR (D₂0, 270MHz) δ 7.97-7.29 (ms, 10H, aromatics), 5.28 (d, IH, Jι,₂ 8.9, H-l), 3.90 (m, IH, H-5, 3), 3.48 (m, 2H, H-2, 4); υmax (KBr) 3416, 1653, 1574, 1420, 1277, 1076, 770 cm-¹; LRMS 423 [M - H]-.

Example 17: (N-(2-Pyrazinoyl)-ff-D-glucopyranosylamine)uronic acid

[α]_D= -11.6° (c 0.25, H₂0); NMR (D₂0, 270MHz) δ 7.95-7.40 (ms, 3H, aromatics), 5.25 (d, IH, J_u 8.6, H-l), 3.88 (d, IH, J ₅ 9.5, H-5), 3.66 (m, 3H, H-

2- 4); ¹³C NMR (D₂0, 300MHz): 175.4 (C=0), 169.2 (C=0), 150.5, 147.1 (2C, each d, aromatic CH), 146.8 (s, aromatic C), 146.3 (d, aromatic CH), 82.3, 79.2, 78.8, 74.2, 74.0 (5C, each d, C-1-5); υmax (KBr) 3453, 3253, 2917, 1790, 1659, 1562, 1368, 1239, 1065, 1065, 1041 cm-¹; LRMS 298 [M - H]-.

Example 18: (N-(2-Thiophenoyl)-/^D-glucopyranosylamine)uronic acid

[ ] = -21.1° (c 0.21, H₂0); NMR (D₂0, 270MHz): δ 7.79-7.15 (ms, 3H, aromatics), 5.15 (d, IH, Jι,₂ 8.4, H-l), 3.82 (d, IH, J₄,₅ 9, H-5), 3.56 (m, 3H, H-2, 3, 4); ¹³C NMR (D₂0, 300MHz): δ 178.4 (s, C=0), 168.0 (s, C=0), 139.1 (s, aromatic), 135.4, 133.5, 131.0 (3C, each d, each aromatic), 82.4, 80.9, 78.9, 74.5, 74.4 (5C, each d, C-1-5); υ_maχ (KBr) 3400, 2928, 1913, 1729, 1655, 1614, 1545, 1419, 1296, 1089, 1024 cm-¹; LRMS 302 [M - H]-.

Example 19: (N-(2-Pyridιne-4-carbonyl)- ^D-glucopyranosylamme)uronic acid

NMR (D₂0, 270MHz) δ 8.63 (s, 2H, aromatic-H), 7.71 (d, 2H, aromatic-H) 5.19 (d, IH, J_W 8.2, H-l), 3.83 (d, IH, J ,₅ 9.1, H-5), 3.55 (m, 3H, H-2, 3, 4): ³C

NMR (D₂0, 300MHz) δ 178.4 (s, C=0), 172.2 (s, C=0), 152.3 (2C, each d, each aromatic CH), 144.1 (s, aromatic C), 124.7 (d, aromatic CH), 82.3, 81.0, 79.0, 74.5, 74.4 (5C, each d, C-1-5); υ_maχ (KBr) 3401 (OH), 2960, 1889, 1634, 1551, 1418, 1300, 1087, 1027 cm-¹; LRMS 297 [M - H]-.

Example 20: (N-(2-Chloro-4-nitrobenzoyl)-^-D- glucopyranosylamine)uronic acid

[O]Ό= -4.8° (c 0.79, H₂0); Η NMR (D₂0, 270MHz) δ 8.42-7.57 (ms, 3H, aromatics), 5.19 (d, IH, J_u 9, H-l), 3.86 (d, IH, J₄,s 9.3, H-5), 3.55 (m, 3H, H-

2-4); υ_max (KBr) 3435, 2358, 1675, 1578, 1420, 1352, 1084, 640 cm-¹; LRMS 394 [M - H]-. Example 21: [N-(3,4-Difluorobenzoyl)-/^D-glucopyranuronosylamine] uronic acid

[ ]_D= -17.7° (c 0.62, MeOH); -NMR (D₂0, 270 MHz): δ 7.76 (m, IH, Ar-H), 7.65 (m, IH, Ar-H), 7.34 (m, IH, Ar-H), 5.16 (d, IH, Jι,₂ 8.0, H-l), 3.82 (d, IH,

/₄,₅ 9.3, H-5), 3.56 (m, 3H, H-2, H-3 and H-4); ³C NMR (D₂0, 300MHz) δ 177.6 (C=0), 172.3 (C=0), 156.6, 153.6 (2 x aromatic CF), 132.9 (s, aromatic C), 127.6 (d, aromatic CH), 120.7, 120.1 (2C, each d, aromatic CH), 82.6, 80.4, 79.1, 74.4, 74.4 (5C, each d, C-1-5); υmax (KBr) 3412, 2940, 1759, 1662, 1597, 1512, 1431, 1378, 1228, 1074 cm-¹ ; HRMS: Found [M - H]-, 332.0576. Cι₃Hι₂ F2NO7 requires 332.0582.

Example 22: [N-(Naphthalene-2-carbonyl)-/^D-glucopyτanuronosylamine] uronic acid

[CC]D= +47.5° (c 0.18, H2O); Η-NMR (D₂0, 300 MHz): δ 8.37 (s, IH, Ar-H), 7.97 (m, 3H, Ar-H), 7.81 (m, IH, Ar-H), 7.60 (m, 2H, Ar-H), 5.20 (d, IH, ]ι,₂ 8.8, H-l), 3.83 (d, IH, /₄₅ 9.5, H-5), 3.54 (m, 3H, H-2, H-3 and H-4); υ_maχ (KBr)

3447, 2925, 1658, 1596, 1422, 1077, 1024. Example 23: [N-(lH-indole-2-carbonyl)- ?-D-glucopyranuronosylamine] uronic acid

[CC]D= +5.0° (c 0.026, MeOH); -NMR (D₂0, 300 MHz): δ 7.73 (m, 1 H, Ar-H), 7.55 (m, 1 H, Ar-H), 7.35 (m, 1 H, Ar-H), 7.26 (s, 1 H, Ar-H), 5.20 (d, 1 H, /ι₂ 8.9, H-l), 3.83 (d, 1 H, / 9.3, H-5), 3.60 (m, 3 H, H-2, H-3 and H-4); ¹³C NMR (300 MHz, D₂0) 178.5, 167.2 (each s, each C=0), 139.8, 132.6,129.8, (s, aromatic C), 128.0, 125.2, 123.58, (each d, each aromatic CH), 115.2 (s, alkene- C) 108.3 (d, aromatic C), 82.32 (d, C-l), 80.9, 79.1, 74.6, 74.5 (each d, C-2-5); υmax (KBr) 3421, 2936, 2359, 1975, 1623, 1559, 1410, 1224, 1072, 882 cm-¹; LRMS 335 [M-H]-.

Example 25: (N-3(lH-indol-3-yl)-ρropionyl)-β-D- glucopyranosylamine)uronic acid

M.p = 41-44°C, [α]_D= -10.26° (c 1.49, H₂0); -NMR δ (300 MHz, D₂0) 7.74 (d, IH, J 7.3, indole-H), 7.55 (d, IH, J 9.2, indole-H), 7.48-7.06 (m, 3H, indole-3H), 5.0 (d, IH, J 9.0, H-l), 3.71 (d, IH, J 9.0, H-5), 3.68-3.49 (ms, 2H, H-3, H-4), 3.43 (apt t, IH, J 9.0, H-2), 3.15 (t, 2H, J 7.3, CH₂), 2.75 (t, 2H, J 7.5, CH₂); ¹³C NMR (300 MHz, D₂0) 181.7, 177.8 (each s, each OO), 136.4, 126.8 (s, aromatic C), 123.3, 122.1, 119.3, 118.8, 113.5 (each d, each aromatic CH), 112.2 (s, alkene- C), 79.3 (d, C-l), 78.1, 76.5, 72.0, 71.9 (each d, C-2-5), 36.9, 20.8 (each t, CH₂); υ_ma_x (KBr) 3420, 2920, 2358, 1578, 1417, 1076, 893, 512 cm-¹; LRMS 363 [M-H]-.

Example 26: (N-(4-biphenylacetyl-2,3,4-tri-O-acetyl-β-D -glucopyranosyl amine)uronic acid 36

[α]_D= -76.3° (c 0.08, MeOH); NMR δ (300 MHz, D₂0) 7.71-7.40 (ms, 9H,

Ar-H), 5.0 (d, IH, J 9.0, H-l), 3.85 (d, IH, J 9.0, H-5), 3.73 (s, CH₂), 3.73-3.51 (ms, 2H, H-3, H-4), 3.44 (apt t, IH, J 9, H-2); ¹³C NMR (300 MHz, D₂0) 178.2, 177.2 (each s, each OO), 143.0, 142.3, 136.6 (s, aromatic C), 132.6, 131.9, 130.4, 130.0, 129.6 (each d, each aromatic CH), 82.2 (d, C-l), 80.0, 79.1, 74.5, 74.4 (each d, C-2-5), 44.7(t, CH₂); υ_maχ (KBr) 3365, 2912, 1849, 1658, 1449, 1603,

1449, 1024 cm-¹; LRMS 386 [M-H] - .

Example 27: N-(3-Methyl-4-oxo-2-phenyI-4H-chromene-8-carbonyl)-β-D- glucopyranuronosylamine)uronic acid

[α]_D= -7.6° (c 0.05, MeOH); H NMR δ (300 MHz, D₂0) 8.24 (d, IH, J 6.5, Ar- H), 8.07 (d, IH, J 8.7, aromatic-H), 7.82-7.56 (m, 2H, Ar-H), 7.56-7.51 (ms, 4H, Ar-H), 5.16 (d, IH, J 9.0, H-l), 3.79 (d, IH, J 9.5, H-5), 3.81-3.47 (ms, 2H, H-3, H-4), 3.36 (apt t, IH, J 9, H-2), 2.09 (s, 3H, CH₃); ¹³C NMR (300 MHz, D₂0) 183.1, 170.5, 165.3 (each s, each OO), 155.5, (s, aromatic C), 137.2 (d, aromatic CH), 134.8 (s, aromatic C), 133.9, 131.9, 131.8, 131.4, 131.3, 131.3, 128.5 (each d, each aromatic CH), 126.8, 124.6, 120.2, 118.0 (s, aromatic C), 82.5 (d, C-l), 79.3, 79.0, 74.6, 74.0 (each d, C-2-5), 13.8 (q, CH₃); υ_ma* (KBr) 3435, 2921, 2490, 1727, 1619, 1443, 1216, 1033, 836, 720 cm-¹; LRMS 454 [M-H]-.

Example 28: N,N'-Di( M glucopyranuronosyl)-terephthalamide

R 0.69 (MeOH); [α]_D +15.0° (c 0.04, H₂0); mp 130-132°C; -NMR: δ (300 MHz, D₂0) 7.99 (s, 2H, aromatic H), 5.33 (d, IH, /ι,₂ 8.7, H-l), 4.10 (d, IH, / 9.2, H-5), 3.59-3.77 (m, 3H, H-2-4); ¹³C-NMR: δ (D₂0) 174.2 (s, COOH),

171.0 (s, OO, amide), 136.7 (s, aromatic C), 128.2 (d, aromatic C), 80.1 , 77.4, 76.5, 71.8, 71.7 (each d); υ_max (KBr) 3437, 2929, 1792, 1645, 1550, 1442, 1234, 1063 cm-¹; LRMS-ES negative ion: Found 515.0 [M-2H]-, Example 29: N-( ^D-glucopyranosyl)benzamide

R 0.18 (MeOH: EtOAc, 1:4); [ ]_D +45.0° (c 0.04, H₂0); p 218-220°C; - NMR: a (300 MHz, D₂0); 7.91 (d, 2H, / 7.2, aromatic H), 7.73 (apt t, IH, / 7.2, aromatic H), 7.62 (apt t, 2H, / 7.2, aromatic H), 5.27 (d, IH, /ι,₂ 9.3, H-l),

3.98 (dd, IH, J_6a,5 2.2, /_6a,6b 12.4, H-6a), 3.84 (dd, IH, J₆b,5 5.1, /_6b,6_a 12.3, H- 6b), 3.65-3.73 (overlapping signals, 3H, H-3, H-4, H-5), 3.57 (apt t, IH, J₂,ι = J₂,3 = 9.3, H-2); ⁱsC-NM : δ (D₂0) 172.2 (s, OO), 133.1 (s, aromatic C), 133.0, 129.1, 127.7 (each d, aromatic C), 80.2 (d, C-l), 77.9, 76.8, 72.0, 69.5 (each d), 60.8 (t); υmax (KBr) 3400, 2856, 1663, 1526, 1291, 1090 cm-¹; HRMS-FAB:

Found 306.0954 [M+Na]⁺, required 306.0952.

Example 31 (N-(Tetrahydro-furan-2carbonyl)-_>-?-D-glucopyranuronosyl- amine)uronic acid.

[_O]_Ό= -18.7° (c=0.08, MeOH). H NMR δ (300 MHz, D₂0) 4.95 (d, IH, J 8.0,

H-l), 4.42 (d, IH, J 8.3, H-7*), 3.95 (dd, IH, J 6.0, 14.0, H-l*), 3.85 (dd, IH, J 6.5, 4.0, H-2*), 3.75 (d, IH, J 9.0, H-5), 3.50 (ms, 3H, H-2, H-3, H-4), 2.28 (m,

IH, H-7*), 2.02- 1.84 (ms, 3H, H-3*, H-4*, H-5*); ¹³C NMR (300 MHz, D₂0)

180.3, 178.5 (each s, each OO), 81.7 (d, C-l), 80.8 (d, C-5), 80.5 (s), 79.0, 78.9, 74.5, 74.4 (each d, C-2-4), 72.7, 33.0, 27.8 (each t, CH₂); υ_max (KBr) 3429, 2921, 2343, 1536, 1424, 1092, 668 cm-¹; LRMS 290 [M-H] -.

Example 34: 1-O-Phenyl-α-D-glucopyranuronic acid

2,3,4-Tri-0-acetyl-β-D-glucopyranurono-6,l-lactone (0.80 g, 2.65 mmol)

(Takeda et al. (1982) Carbohydr. Res. 106, 175-192) was dissolved in dry dichloromethane (10 mL) under N2 atmosphere. SnCk (0.15 mL, 0.5 eq) and phenol (0.78 g, 2.5 eq) were added. The reaction was stirred overnight, diluted with dichloromethane, stirred again in presence of saturated aqueous NaHCθ3 (20 mL) for 1 h and filtrated through filter paper. When organic and aqueous layers were separated, the majority of carbohydrate product was found to be in the aqueous layer, therefore the latter was freeze-dried to give a white solid (1.336 g). NMR analysis showed this to be mainly the sodium salt contaminated by some phenol and other inorganic salts. The solid was then redissolved in water and the pH adjusted to 2 adding Amberlite IR-120: the product could be extracted in EtOAc, dried on MgS0 , concentrated and recrystallized from dichloromethane/ pet. ether to give 2,3,4-tri-O-acetyl-l-O- phenyl-α-D-glucopyranuronic acid (0.672 g, 64%); -NMR (300 MHz, CDC1₃): δ 8.15 (br s, IH, COOH), 7.30 (dd, 2H, /_a, 8.9, J_b,_c 7.1, Ar H-b and H- b'), 7.09 (m, 3H, Ar H-a, H-a' and H-c), 5.81 (d, IH, /ι,₂3.5, H-l), 5.76 (apt t,

IH, /₂,₃ 10.0, J_3/ 9.5, H-3), 5.31 (dd, IH, J ,₅ 10.1, H-4), 5.07 (dd, IH, H-2), 4.49 (d, IH, H-5), 2.06, 2.05, 2.04 (each s, each 3H, each COCH3); ¹³C-NMR (300 MHz, CDCI₃): δ 171.1 (s, COOH), 170.6, 170.5, 170.3 (each s, each COCH3), 156.3 (s, Ar C ipso), 130.0 (d, Ar C-b and C-b'), 123.7 (d, Ar C-c), 116.9 (d, Ar C-a and C-a'), 94.7 (d, C-l), 70.4, 69.7, 69.6, 68.5 (each d, C-2-C-5), 20.9, 20.8,

20.7 (each q, each COCH3). This intermediate (0.092 g, 0.232 mmol) was suspended in LiOH 0.1N (10 mL) in MeOH/ water/ THF (2.5/1.0/0.5) at O⁰C (ice-bath) and stirred for 2 h and the mixture was diluted with water. After adjusting the pH to 2 using Amberlite IR-120, the solvents were evaporated in vacuo giving example 34 (0.063 mg, 100%); -NMR (300 MHz, D₂0): δ 7.31 (dd, 2H, J_a,_b 8.3, J_b,c 7.4, Ar H-b and H-b'), 7.07 (m, 3H, Ar H-a, H-a' and

H-c), 5.61 (d, IH, Jι,23.7, H-l), 4.14 (d, IH, J₄₅ 10.1, H-5), 3.91 (apt t, IH, /₂₃ 9.6, J₃,49.4, H-3), 3.72 (dd, IH, H-2), 3.58 (dd, IH, H-4); ¹³C-NMR (300 MHz, D₂0): δ 174.4 (s, COOH), 156.1 (s, Ar C ipso), 130.2 (d, Ar C-b and C-b'), 123.6 (d, Ar C-c), 117.4 (d, Ar C-a and C-a'), 97.3 (d, C-l), 72.9, 72.2, 71.8, 71.0 (each d, C-2-C-5).

Analytical and spectroscopic data for intermediates

Intermediate 6a: (N-(Benzoyl)-2,3,4-tri-O-acetyl-y£-D- glucopyranosylamine)uronic acid, methyl ester

Benzoyl chloride (0.23 mL, 2 mmol), intermediate 5 (0.36 g, 1 mmol) and triphenylphosphine (0.34 g, 1.3 mmol) were treated as described above to afford the title compound (0.27 g, 62%); [ ]²⁰ _D +3.6° (c 1.0, CHC1₃); Η NMR (CDCI3, 300MHz) δ 7.78-7.41 (ms, 5H, aromatic) 7.17 (d, IH, / 9.4, NH), 5.48 (m, 2H, H-l, 3), 5.19 (apt t, IH, J₃, 9.6, H-4), 5.11 (apt t, IH, /₂,ι 9.6, H-2), 4.25

(d, IH, J₄,5 9.9, H-5), 3.72 (s, 3H, OMe), 2.06, 2.05, 2.04 (3s, each 3H, 3xOAc); C NMR (CDC13, 300MHz) δ 171.6, 169.9, 169.8, 167.5, 167.4 (each s, each OO), 132.7-127.5 (5C, each d, aromatic), 130.4 (s), 78.7 (d, C-l), 74.1 (d, C-5), 71.8 (d, C-3), 70.5 (d, C-2), 69.8 (d, C-4), 52.9 (q, OMe), 20.7, 20.6, 20.5 (3xOAc, each q); IR (liquid film): υ, 2350, 1753 (OO), 1668 (OO), 1536, 1370, 1225, 1038 cm-¹; MS: Calcd. for C₂₀H₂6N₂Oιo 438.1400 (M+NH4); found 438.1399.

Intermediate 7: Succinimidoethyl-2,3,4-tri-O-acetyl-β-D-glucopyranuronic acid, methyl ester.

N-(2-Hydroxyemyl)succirιimide (200 mg, 1 mmol) and intermediate 2 (0.06 g, 1.5 mmol) were reacted according to method A to give the title compound (0.18 g, 40 %): m.p. 195-200 °C; [α] -21.8 (c 0.096, CHCI3); NMR δ (300 MHz, CDCI3) 5.22 (overlapping signals, 2H, H-3, H-4), 4.94 (apt t, IH, J 7.0, H-2), 4.58 (d, IH, J 7.0, H-l), 4.03 (m, 2H, OCH(H), H-5), 3.70-3.08 (ms, 6H,

OCH3, CH2N, CH(H)0), 2.07 (s, 4H, CH₂CH₂00), 2.01, 2.04, 2.06 (each s, each 3H, each OAc); ¹³C NMR δ (CDCI3) 177.1 (s, OO, methyl ester), 171.1, 170.1, 169.3 (each s, each OO), lOO.ld, C-l), 72.5, 72.0 , 71.3, 69.3 (each d, C- 2-5), 65.5 (t, C-6), 52.9 (q, OCH3), 38.1 (t, CH₂), 28.2 (t, CH₂), 20.7, 20.6, 20.5 (each q, each OAc); IR (KBr) 2982, 1759, 1705, 1395, 1273, 748 cm-¹. Anal.

Calcd for G9H25O12N: C, 49.67, H, 5.70, N, 3.04. Found C, 49.59, H, 5.61, N, 3.04. MS Calcd for M+Na 482.1292; Found 482.1292.

Intermediate 8: Phthalimidoethyl-2,3,4-tri-O-acetyl-β-D-glucopyranuronic acid, methyl ester.

N-(2-Hydroxyethyl)phthalimide (200 mg, 1 mmol) and intermediate 2 (0.6 g, 1.5 mmol) were reacted according to method A to give the title compound (0.26 g, 52 %): m.p. 135-135 °C; [ ]_D -30.2 (c 0.1, CHC1₃); NMR δ (300 MHz,

CDCI3) 7.87-7.83 (m, 4H, aromatic-H), 5.21 (overlapping signals, 2H, H-3 and H-4), 4.95 (dd, IH, J 7.5 and 6, H-2), 4.59 (d, IH, J 7.5, H-l), 4.16-3.78 (ms, 5H, H-5, OCH₂CH₂), 3.72 (s, 3H, OCH3), 1.86, 1.98, 2.00, (each s, each 3H, OAc); ³C NMR δ (CDCI3) 170.0 169.4, 169.2, 168.2, 169.0, 166.1 ((each s, each OO) ), 134.2 (d, aromatic-CH), 132.2 (s, aromatic C), 123.2 (d, aromatic CH), 100.4

(d, C-l), 72.6 72.1, 71.1, 69.5 (each d, C-2-5). IR (KBr) 2389, 1759, 1694, 1362, 1217, 1072 cm-¹. MS Calcd for M+Na 530.1274; Found 530.1278.

Intermediate 9: (l,4-Dioxaspirol[4,5] dec-2-yl)-2,3,4-tri-O-acetyl-β-D- glucopyranuronic acid, methyl ester.

(+)l,4-Dioxaspirol[4,5]decan-2-methanol (200 mg, 1.16 mmol) was reacted with intermediate 2 (0.60 g, 1.5 mmol) according to method A to give the title compound (mixture of diastereoisomers obtained, 0.23 g, 41%); [CX]D -21.75 (c

0.30, CHCI3); NMR δ (300 MHz, CDCI3) 5.24-5.21 (ms, 2H, H-3, H-4), 5.00-

5.01 (ms, IH, H-2), 4.67 (d, IH, J 8.0, H-l), 4.24 (m, IH), 4.17-3.95 ( , 2H),

3.92-3.60 (m, 6H, CH, OCH₃, CH₂), 2.04, 2.03, 2.01 (each s, each 3H, OAc),

1.26 ( , 10H, CH₂); ¹³C NMR δ (CDCI3) 170.3 169.5 , 169.5 (2s) , 169.4, 167.4 , 167.4 (each OO), 110.4, 110.1, 101.2 (each s), 74.3, 74.1, 72.9, 72.8, 72.3, 72.3, 71.4, 71.3 (each d), 71.3 (t), 69.7, 69.6 (each d), 69.6, 66.6, 66.0 (each t), 53.1 (q), 36.7, 36.5 , 35.1, 34.9, 35.3, 24.2, 24.0, 24.0(each t), 21.2,, 20.8, 20.7 (each q, OAc); IR (KBr) 3054, 2305, 1758, 1438, 1265, 1220, 738 cm-¹. Anal. Calcd for C₂₂H₃₂Oι₂: C, 54.01, H, 6.59. Found C, 53.85, H, 6.56. HRMS Calcd for M+Na 511.1791; Found 511.1805.

Intermediate 10: (3-Benzoylphenyl)-2,3,4-tri-O-acetyl-β-D- glucopyranuronic acid, methyl ester.

3-Hydroxybenzophenone (200 mg, 1 mmol) was reacted with intermediate 4 (0.47 g, lmmol) according to method B to give the title compound (0.26 g, 52 %): m.p. 55-60 °C; [α]_D -36.7 (c 0.12, CHCls); NMR δ (300 MHz, CDCI3) 7.85-7.20 (m, 9H, aromatic-H), 5.23-5.50 (overlapping signals, 4H, H-l-4), 4.11-4.24 (ms, IH, H-5), 3.71 (s, 3H, OCH3), 2.03 (s, 9H, OAc); ³C NMR δ

(CDCI3) 196.0 (s, benzophenone OO), 170.2 , 169.5, 169.4, 167.0 (each s, each OO), 156.7 , 39.4, 137.5 (each s, each aromatic C), 132.9, 130.3, 129.8, 128.6, 125.5, 121.5, 118.2 (each d, each aromatic CH), 99.1 (d, C-l), 72.8, 72.1, 71.3, 69.3 (each d, C-2-5), 53.2 (q, OCH₃), 20.80, 20.70 (each q, OAc); IR (KBr) 1758, 1659, 1223 cm"¹; HRMS Calcd M+Na 537.1373; Found 537.1374.

Intermediate 11: (N-(2-Biρhenylcarbonyl)-2,3,4-tri-O-acety.l- ?-D- glucopyranosylamine)uronic acid, methyl ester

2-Biphenylcarbonyl chloride (1.1 g, 4.9 mmol), prepared in method D, intermediate 5 (0.6 g, 1.6 mmol) and triphenylphosphine (0.65 g, 2.4 mmol) were treated as described in method C to afford the title compound (0.34 g, 40%); [ ]²⁰D +3.76° (c 0.186, CHCls); Η NMR (CDCI3, 300MHz) δ 7.39-7.26

(ms, 9H, aromatic), 6.42 (d, IH, / 9.4, NH), 5.30 (m, 2H, H-l, 3), 5.08 (apt t, IH, J₃,49.9, H-4), 4.76 (apt t, IH, /ι,₂ 9.5, H-2), 4.1 (d, IH, /₄₅ 9.9, H-5), 3.73 (s, 3H, OMe), 2.00, 1.99, 1.92 (3s, each 3H, 3xOAc); ³C NMR (CDCI3, 270MHz) δ 172.9, 170.6, 170.1, 169.8, 167.1 (each s, each OO), 142.9, 141.1, 134.0 (3C, each s, each aromatic CH), 131.7-127.0 (9C, each d, aromatic), 78.0 (d, C-l),

73.8, 72.0, 70.0, 69.5 (each d, C-2-5), 52.8 (q, OMe), 20.6, 20.5 (2 signals) (3xOAc, each q); IR (liquid film): υ 3328, 3065, 2635, 2120, 1754 (OO), 1527, 1372, 1243, 1038, 749 cm-¹; HRMS-CI (M/Z): [M+NIiJ Calcd. For C₂₆H3iN₂Oιo 514.1713; found 514.1712.

Intermediate 12: (N-(2-phenylquinoline-4-carbonyl)-2,3,4-tri-O-acetyl-/?- D-glucopyranosylamine)uronic acid, methyl ester

2-Phenylquinoline-4-carbonyl chloride (1.32 g, 4.8 mmol), prepared by method D, intermediate 5 (0.59 g, 1.6 mmol) and triphenylphosphine (0.65 g, 2.4 mmol) were treated as described in method C to afford the title compound (0.62 g, 66%); [ ] ⁰ _D +20° (c 0.95, CHCls); NMR (CDCI3, 300MHz) δ 8.34-7.29 (ms, 9H, aromatic), 6.87 (d, IH, / 9.1, NH), 5.64(apt t, IH, fa 9.4, H-3), 5.48 (apt t, IH, H-2), 5.13 (m, 2H, H-l, 4), 4.27 (d, IH, fa 9.9, H- 5), 3.71 (s, 3H, OMe), 2.05, 2.04, 2.03 (2 signals) (3s, each 3H, 3xOAc); ¹³C

NMR (CDCI3, 270MHz) δ 170.9, 169.8 , 169.6, 169.4, 167.6 (each s, each OO), 156.5, 148.6, 140.4 (3c, each s, each aromatic CH), 130.3-116.7 (10C, each d, aromatic), 122.9 (s), 78.1 (d, C-l), 73.9, 71.9, 70.6, 69.5 (each d, C-2-5), 52.9 (q, OMe), 20.7, 20.6, 20.5 (3xOAc, each q); IR (liquid film): υ 3280 (NH), 3053, 2956, 2122, 1748 (OO), 1660 (OO), 1530, 1373, 1217, 1066, 890, 770, 587 cm-¹;

HRMS-CI (M/Z): [M+NH4] Calcd. For C₂9H₃₂OιoN₃ 587.1641; found 587.1637.

Intermediate 13: (N-(4-Chloroρhenylacetyl)-2_/3,4-tri-O-acetyI-β-D- glucopyranosylamine)uronic acid, methyl ester.

Treatment of 4-chlorophenylacetyl chloride (1.5 mmol, 1.5 g), prepared by method D, and intermediate 5 (1.52 g, 4 mmol) as described in method C gave intermediate 13 (99 mg, 5 %): m.p. 197 °C; [a] -0.58 (c 1.2, CHCI3); - NMR δ (300 MHz, CDCI3) 7.13-7.35 (ms, 4H, aromatic-H), 6.35 (d, IH, J 10.0,

NH), 5.34 (IH, apt. t, J 10.0, H-3), 5.23 (IH, apt. t , J 10.0, H-l), 5.11 (apt. t, J 10.0, H-4), 4.86 (apt. t, J 10.0, H-2), 4.14 (d, IH, J 10.0, H-5), 3.72 (s, 3H, OCH₃), 3.49 (AB d, 2H, J 15.0, CH₂), 2.02, 2.00, 1.87 (each s, each 3H, each OAc); IR (KBr) 3304, 1745, 1669 cm-¹. Anal. Calcd for C₂ιH₂₄ClNOιo: C, 51.91, H, 4.98, N, 2.88. Found C, 51.66, H, 4.91, N, 2.67; MS 486.0 (M+H)⁺ Intermediate 14: (N-(3-methyl-2-but-2-enoly)-2,3,4-tri- -acetyl-β-D- glucopyranosylamine)uronic acid, methyl ester.

Treatment of 3-methyl-2-but-2-enoyl chloride (0.44 g, 3.8 mmol), prepared by method D, with intermediate 5 (0.69 g, 1.9 mmol) as described in method C gave the title compoimd (0.27 g, 33 %): m.p. 85-95 °C; [α]_D +1.6 (c 0.064, CHCls); NMR δ (300 MHz, CDCI3) 6.32 (d, IH, J 9.5, NH), 5.50 (s, IH, alkene CH), 5.39 (apt. t, IH, J 9.5, H-3), 5.34 (apt. t, IH, J 9.5, H-4), 4.97 (apt. t, IH, J 9.5, H-3), 4.17 (d, IH, J 10.0, H-5), 3.72 (s, 3H, OCH3), 2.15 (d, 3H, J 1.0, CH3), 2.03 (s, 9H, OAc), 1.85 (d, 3H, J 1.0, CH3); ¹³C NMR δ (300 MHz, CDCI3)

171.2, 169.9, 169.8, 167.5, 166.7 (each s, each OO), 155.8 (s, alkene C), 117.4 (d, alkene CH), 78.3 (d, C-l), 72.3, 70.5, 70.0 (each d, C-2-5), 53.1 (q, OCH3), 27.6, 20.9 (each q, each CH3=C), 20.8, 20.7, 20.3 (each q, each OAc); IR (KBr) 3330, 1753, 1644, 1535, 1378 cm-¹; HRMS Calcd (M+H)⁺ 416.1557; Found 416.1557.

Intermediate 15: (N-(3-furan-2-yl-acryloyl)-2,3,4-tri-O-acetyl-β-D-

;lucoρyranosylamine)uronic acid, methyl ester.

Treatment of 3-furan-2-ylacryloyl chloride (1.4 g, 9 mmol), prepared by method D, and intermediate 5 (1.61 g, 4.5 mmol) as described by method C gave intermediate 15 (0.82 g, 40 %): m.p. 205 °C; [α]_D +1.37 (c 0.50, CHCI3); -NMR δ (300 MHz, CDCI3) 7.36 (ms, 2H, furan-H alkene-H), 6.65 (d, IH, J 9.0, NH), 6.53 (d, IH, J 3.0, furan-H), 6.38 (d, IH, J 1.5, furan-H), 6.19 (d, IH, J 15.0, alkene-H), 5.30-5.44 (ms, 2H, H-3 and H-l); 5.11 (apt. t, IH, J 10.0, H-4), 4.98 (apt. t, IH, J 9.5, H-2), 4.16 (d, IH, J 10.0, H-5), 3.65 (s, 3H, OCH₃), 1.97 (s, 9H, OAc); ³C NMR δ (CDCI3) 171.1, 170.0, 169.8, 167.5,166.3 (each s, OO), 151.10 (s, furan C), 130.1, 117.2, 115.2, 112.5(each d, furan and alkene CH),

78.5 (d, C-l), 74.2, 72.3, 70.5, 69.9(each d, C-2-5), 53.1 (q, OCH3), 20.8, 20.7,

20.6 (each s, each OAc); IR (KBr) 3340, 2898, 1665, 1633, 1228, 1038, 745cm-¹. Anal. Calcd for CajHzjNOπ: C, 52.98, H, 5.11, N, 3.09. Found C, 53.98, H, 5.08, N, 2.97; MS 476.0 (M+Na)

Intermediate 16: (N-(4-Biphenylacetyl)-2,3,4-tri-O-acetyl-β-D- glucopyranosylamine)uronic acid, methyl ester.

Treatment of 4-biphenylacetyl chloride (1.58 g, 7 mmol), prepared by method E, and intermediate 5 (1.25 g, 3.5 mmol) as described in method C gave intermediate 16 (0.59 g, 32 %); m.p. 205 °C; [ ]_D -15 (c 0.64, CHCI3); NMR δ (300 MHz, CDCI3) 6.41 (d, IH, J 9.5, NH), 5.35 (apt. t, IH, J 9.5, H-2), 5.26

(apt. t, IH, J 9.5, H-l), 5.12 (apt. t, IH, J 9.5, H-4), 4.88 (apt. t, J 9.8, H-3), 4.16 (d, IH, J 10.0, H-5), 3.72 (s, 3H, OCH3), 3.72 (AB d, 2H, J 15.0, CH₂), 2.01, 1.99, 183 (each s, each 3H, OAc); ¹³C NMR δ (300 MHz, CDC1₃) 171.5, 170.7, 169.8, 169.7, 167.3(each s, each OO), 140.0, 132.9, 129.8 (each s, aromatic C), 129.0, 128.0, 127.6, 127.2(each d, aromatic CH), 78.4 (d, C-l), 74.2, 72.0, 170.1, 70.0

(each d, C-2-7), 43.6 (t), 20.6, 20.7, 20.5 (each q); IR (KBr) 3076, 2954, 1752, 1695, 1374, 1221, 750cm-¹, HRMS Calcd (M+H) 528.1870; Found 528.1871 Intermediate 17: (N-(2-Methylpentanoyl)-2,3,4-tri-O-acetyl-β-D- glucopyranosylamine)uronic acid, methyl ester.

Treatment of 2-methylpentanoyl chloride (0.94 g, 6.0 mmol), prepared by method E, and intermediate 5 (1.12 g, 3.0 mmol) as described in method C gave intermediate 17 (0.54 g, 42 %, 1:1 mixture of diastereoisomers): m.p. 151-160 °C; [ ]_D +18.2 (c 0.13, CHC1₃); Η NMR δ (300 MHz, CDCI3) 6.35 (apt. t, 1H)NH), 5.44-5.28 (ms, 2H, H-l and H-3), 5.14 (apt. t, IH, J 9.5, H-4), 4.97 (t, IH, J 9.6, H-2), 4.16 (d, IH, J 10.0, H-5), 3.73 (s, 3H, OCH₃), 2.20 (m, 2H, CH₂), 2.03 (s, 9H, OAc), 1.67-0.91 (ms, 10H); ¹³C NMR δ (CDCI3) 177.1 , 177.1, 171.1,

171.0, 169.9, 169.7, 167.4 (each s, each OO), 78.2, 78.1 (each d, each C-l), 74.3, 74.2, 72.1, 72.1, 70.6, 70.0 (each d, C-2-5), 53.1 (q, OCH3), 41.5, 41.4 (each d), 36.5, 35.7 (each t), 20.8, 20.7(each q), 20.620.6 (each t), 17.7, 17.3, 14.2, 14.1 (each q), IR (KBr) 3338, 2955, 1743, 1666, 1530, 1351, 773 cm-¹, HRMS Calcd (M+H) 432.1870; Found 432.1865.

Intermediate 18: (N-2-cycloproρanecarbonyl)-2,3,4-tri-O-acetyl-β-D- glucopyranosylamine)uronic acid, methyl ester.

Treatment of cyclopropanecarbonyl chloride (1.0 g, 4.0 mmol), prepared by method E, and intermediate 5 (1.72 g, 4.8 mmol) as described in method C gave intermediate 18 (0.91 g, 48). m.p.151-160 °C; [α]_D 0.00 (c 0.25, CHCI3); NMR δ (300 MHz, CDCI3) 6.71 (d, IH, J 9.5, NH), 5.42-5.28 (ms, 2H, H-3 and H-l), 5.13 (apt. t, IH, J 9.5, H-4), 4.98 (apt. t, IH, J 9.5, H-2), 4.15 (d, IH, J 9.5, H-5), 3.72 (s, 3H, OCH3), 2.04, 2.03, 2.02, (each s, each 3H, OAc), 1.42-0.74 (ms, 5H, cyclopropyl H), ¹³C NMR δ (300 MHz, CDCI3) 171.1, 169.9, 169.8, 167.5 (each s, each OO), 78.3 (d, C-l), 74.2, 70.5, 70.0 (each d), 53.1 (q, OCH₃), 20.9, 20.8, 20.7 (each q), 15.1, 8.59 (t), 8.39 (t); IR (KBr) 3385, 1762, 1684, 1232, 1190, 754cm-¹. MS Found, 424.0 (M+Na), requires 424.1.

Intermediate 19: (N-(2,4-Dichlorophenoxyaceryl)-2,3, ~tri-O-acetyl-β-D- glucopyranosylamine)uronic acid, methyl ester.

Treatment of 2,4-dichlorophenoxyacetyl chloride (2.07 g, 8.00 mmol), prepared by method E, with intermediate 5 (1.45 g, 4.0 mmol) as described in method C gave intermediate 19 (0.25 g, 11%).; Η NMR δ (300 MHz, CDCI3) 7.70-7.40 (m, 3H, aromatic H and NH), 6.79 (d, IH, J 8.5, aromatic H), 5.45- 5.35 (m, 2H, H-3 and H-l), 5.36 (apt. t, IH, J 9.5, H-4), 5.07 (apt. t, IH, J 9.5, H-2), 4.53 (AB d, 2H, J 15.0, CH₂), 6.79 (d, IH, J 9.0, H-5), 3.73 (s, 3H, OCH3),

2.03, 2.02, 1.95 (each s, each 3H, OAc), ¹³C NMR δ (300 MHz, CDCI3) 170.2, 169.9, 169., 168.6, 167.2 (each s, each OO), 151.7 (s, aromatic C), 130.7, 128.0(each d, each aromatic CH), 128.0, 124.6 (each s, each aromatic C), 76.9 (d, C-l), 74.3, 72.3, 70.1, 69.8 (each d, C-2-5), 68.4 (t, CH₂), 53.2 (q, OCH₃), 20.75, 20.67, 20.6 (each q, OAc); IR (KBr) 3424, 1746, 1683, 1549, 1071, 747cm-¹;

MS 558.0 (M+Na), requires 558.1. Intermediate 20: (N-(2-Pyrazinoyl)-2,3,4-tri- -acetyl-y#-D- glucopyranosylamine)uronic acid methyl ester

2-Pyrazinoyl chloride (0.7 g, 4.9 mmol), intermediate 5 (0.59 g, 1.65 mmol) and triphenylphosphine (0.65 g, 7.35 mmol) were treated as described in method C to afford the title compound (0.14 g, 19%); [ ]²⁰ _D +3.85° (c 0.234, CHCls); NMR (CDCI3, 300MHz) δ 9.39-8.58 (ms, 3H, aromatic), 8.56 (d, IH, / 8.4, NH), 5.52 (apt t, IH, fa 9.5, H-l), 5.47 (apt t, IH, fa 9.5, H-3), 5.23 (apt t, IH, H-4), 5.18 (apt t, IH, H-2), 4.27 (d, IH, fa 9.9, H-5), 3.73 (s, 3H, OMe), 2.05 (2 signals), 1.99 (3s, each 3H, 3xOAc); ¹³C NMR (CDC13, 300MHz):

170.6, 169.9,169.7,167.3, 164.0 (each s, each OO), 148.2, 144.9,143.2 (3C, each d, aromatic), 143.5 (s), 78.2 (d, C-l), 74.4, 72.3, 70.4, 69.9 (each d, C-2-C-5), 53.2 (q, OMe), 20.8, 20.7, 20.7 (3xOAc, each q); IR (liquid film): υ 3371 (NH), 2953, 1757 (OO), 1685 (OO), 1514, 1376, 1259, 1039 cm-¹; MS: Calcd. for [M+NH4] GoHΛOio 440.1305 found 440.1303..

Intermediate 21: (N-(2-Thiophenoyl)-2,3,4-tri-O-acetyl-y5-D- glucopyranosylamine)uronic acid, methyl ester

2-Thiophenoyl chloride (0.5 g, 3.4 mmol), prepared by method D, intermediate 5 (0.41 g, 1.14 mmol) and triphenylphoshine polystyrene (0.5 g, 3.4 mmol) were treated as described in method C to afford the title compound (0.26 g, 53%); [αpo_D -15.7° (c 0.134, CHCI3); NMR (CDCI3, 300MHz) δ 7.88-7.05 (ms, 3H, aromatic), 7.40 (d, IH, / 9, NH), 5.54 (m, 2H, H- 1, 3), 5.19 (apt t, IH, fa 9.5, H-4), 5.09 (apt t, IH, fa 9.5, H-2), 4.27 (d, IH, fa 9.9, H-5), 3.71 (s, 3H, OMe), 2.07, 2.05, 2.04 (3s, each 3H, 3xOAc); ¹³C NMR (CDCI3, 300MHz) δ 171.3, 169.8, 169.3, 167.2, 161.9 (each s, each OO), 137.4 (s, aromatic C), 134.6-127.8 (3C, each d, aromatic CH), 78.5 (d, C-l), 73.7, 71.8, 70.4, 69.6 (each d, C-2-5), 52.9 (q, OMe), 20.7, 20.5, 20.4 (3xOAc, each q); IR

(liquid film): υ3354 (NH), 2955, 2358, 1750 (OO) , 1659 (OO), 1541, 1372, 1232, 1037, 734 cm"¹; HRMS-CI (M/Z): [ +NH4] Calcd. For Q9H25N2O10S 444.0964; found 444.0961.

Intermediate 22: (N-(2-Pyridine-4-carbonyl)-2,3,4-tri-0-acetyl-yff-D- glucopyranosylamine)uronic acid, methyl ester

Pyridine-4-carbonyl chloride (0.7 g, 4.9 mmol), prepared by method D, intermediate 5 (0.59 g, 1.65 mmol) and triphenylphosphine (0.65 g, 2.5 mmol) were treated as described in method C to afford the title compound (0.34 g,

46%); [α]²⁰D +10.95° (c 1.78, CHCI3); NMR (CDCI3, 300MHz): δ 8.75-7.48 (ms, 5H, aromatic), 7.92 (d, IH, / 9.0, NH), 5.51 (m, 2H, H-l, 3), 5.13 (m, 2H, H-2, 4), 4.27 (d, IH, fa 9.9, H-5), 3.71 (s, 3H, OMe), 2.07, 2.06, 2.05 (3s, each 3H, 3xOAc); ¹³C NMR (CDCI3, 270MHz): δ 171.2, 169.7, 169.6, 167.1, 165.7 (each s, each OO), 150.7-121.1 (4C, each d, aromatic CH), 139.9 (s), 78.5 (d,

C-l), 73.9, 71.7, 70.5, 69.6 (each d, C-2-5), 52.9 (q, OMe), 20.7, 20.6, 20.5 (3xOAc, each q); IR (liquid film): υ 3201 (NH), 3029, 2429, 2114, 759 (OO), 1687 (OO), 1552, 1441, 1370, 1219, 1082, 851, 696, 541 cm-¹; HRMS-CI (M/Z): [M+NH ] Calcd. For Cι₉H₂6N₃Oιo 439.1352, Found 439.1349. Intermediate 23: (N-(2-Chloro-4-nitrobenzoyl)-2,3,4-rri-O-acetyl-_Jt -D- glucopyranosylamine)uronic acid, methyl ester

2-Chloro-4-nitrobenzoyl chloride (1.1 g, 4.9 mmol), prepared by method D, intermediate 5 and triphenylphosphine (0.65 g, 2.5 mmol) were treated as described in method C to afford the title compound (0.23 g, 28%); [α]²⁰ _D +22.7° (c 0.132, CHCI3); Η NMR (CDCI3, 300MHz) δ 8.33-7.27 (ms, 3H, aromatic), 7.13 (d, IH, / 9.2, NH), 5.52 (apt t, IH, fa 9.5, H-l), 5.45 (apt t, IH, fa 9.6, H-3), 5.18(apt t, IH, H-4), 5.08 (apt t, IH, H-2), 4.24 (d, IH, fa 9.9, H- 5), 3.75 (s, 3H, OMe), 2.08, 2.05 (2 signals) (3s, each 3H, 3xOAc); ¹ C NMR

(CDCI3, 270MHz) δ 170.9, 170.0, 169.8, 167.4, 165.5 (each s, each OO), 149.4 (s) 139.8 (s), 135.6 (s) 135.2,121.7 (3C, each d, aromatic), 78.3 (d, C-l), 74.3, 72.2, 70.4, 69.7 (each d, C-2-5), 53.2 (q, OMe), 20.8, 20.7, 20.6 (3xOAc, each q); IR (liquid film): υ 3328 (NH), 3104, 2946, 1759 (OO), 1681 (OO), 1533, 1350, 1226, 1040, 892, 740 cm-¹; HRMS-CI (M/Z): [M+NHJ. Calcd. For

C₂₀H25ClN₃Oι₂539.0684; found 539.0685.

Intermediate 24: (N-(3,4-Difluorobenzoyl)-2,3,4-tri-O-aceryl-y£-D- glucopyranosylamine)uronic acid, methyl ester

3,4-Difluorobenzoyl chloride (0.2 g, 2.1 mmol), prepared by method D, intermediate 5 and triphenylphosphine bound polystyrene resin (1.6 g, 2.1 mmol) were treated as described in method C to afford the title compound

(0.19 g, 49%); [CC] ⁰D -19.67° (c 0.122, CHC1₃); NMR (CDCb, 300MHz) δ 7.67-7.21 (ms, 4H, 3H aromatic, NH) 7.29 (d, IH, / 9.2, NH), 5.48 (apt t, 2H,

H-l, 3), 5.17 (apt t, IH, fa 9.5, H-4), 5.10(apt t, IH, fa 9.5, H-2), 4.25 (d, IH, fa 9.9, H-5), 3.72 (s, 3H, OMe), 2.07, 2.05 (2 signals) (3s, each 3H, 3xOAc); ³C

NMR (CDCI3, 270MHz) δ 171.5, 169.8, 169.6, 167.2, 165.2 (each s, each OO),

129.8 (s) 124.0-117.2 (5C, each d, aromatic), 78.6 (d, C-l), 73.8, 71.7, 70.6, 69.6 (each d, C-2-5), 52.9 (q, OMe), 20.7, 20.6, 20.4 (3xOAc, each q); IR (liquid film): υ 3351 (NH), 2959, 2122, 1757 (OO), 1679 (OO), 1512, 1372, 1224,

1040, 894, 736 cm-¹; HRMS-CI (M/Z): [M+NHJ Calcd. For C₂₀H₂₅F₂N₂O₁₀

474.1211; found 474.1212.

Intermediate 25: (^(S-TrifluoromethylbenzoylJ^S^tri-O-acetyl-^D- glucopyranosylamine)uronic acid methyl ester

3-Trifluoromethylbenzoyl chloride (1.115 g, 5.5 mmol), prepared by method

D, intermediate 5 (0.8 g, 2.2 mmol) and triphenylphosphine (0.87 g, 2.9 mmol) were treated as described in method C to afford the title compound

(0.27 g, 62%); [α]²⁰ _D +10.6° (c 0.122, CHCI3); Η NMR (CDCb, 300MHz) δ 8.37-

7.56 (ms, 4H, aromatic) 7.38 (d, IH, / 9.0, NH), 5.51 (m, 2H, H-l, 3), 5.20 (apt t,

IH, fa 9.9, H-4), 5.12 (apt t, IH, fa 9.5, H-2), 4.27 (d, IH, fa 9.9, H-5), 3.73 (s,

3H, OMe), 2.07, 2.05 (2 signals) (3s, each 3H, 3xOAc); ¹³C NMR (CDC ) δ 171.5, 169.9, 169.7, 169.4, 166.1 (each s, each OO), 133.5 (s), 133.4-129.2 (C, each d, aromatic), 78.6 (d, C-l), 73.9, 71.8, 70.6 , 69.6 (each d, C-2-5), 53.0 (q, OMe), 20.7, 20.6, 20.5 (3xOAc, each q); IR (liquid film): υ, 3326 (NH), 2960, 2634, 1755 (OO), 1543, 1330, 1229, 1130, 906, 695 cm-¹; HRMS-CI (M/Z): [M+NH ] found 506.1270. Calcd. For C₂ιH₂₆F₂N₂Oιo 506.1274.

Intermediate 26: (N-(3,5-Dimethylbenzoyl)-2,3,4-tri-O-acetyl- ?-D- glucopyranosylamine)uronic acid, methyl ester

3,5-Dimethyl benzoyl chloride (0.93 g, 5.5 mmol), prepared in method D, intermediate 5 (0.8 g, 2.2 mmol) and triphenylphosphine (0.87 g, 2.9 mmol) were treated as described in method C to afford the title compound (0.56 g,

56%); [α] ⁰D +1.0° (c 0.396, CHCI3); NMR (CDC1₃, 300MHz) δ 7.37-7.15 (ms,

3H, aromatic) 7.21 (d, IH, / 9.4, NH), 5.49 (m, 2H, H-l, 3), 5.16 (m, 2H, H-4,

2), 4.23 (d, IH, fa 9.9, H-5), 3.71 (s, 3H, OMe), 2.33 (s, 6H, 2xCHs), 2.06, 20.4, 2.03 (3s, each 3H, 3xOAc); ¹³C NMR (CDCI3) δ 170.9, 169.8, 169.5, 167.6, 167.1

(each s, each OO), 138.1-125.0 (6C, 4d, 2s, aromatic CH), 78.2 (d, C-l), 73.6,

71.9, 70.4 , 69.5 (each d, C-2-5), 52.7 (q, OMe), 20.5 (2 signals), 20.3 (3xOAc, each q); IR (liquid film): υ 3353 (NH), 2959, 2128, 1870, 1754 (OO), 1528,

1373, 1229, 1035, 887 cm-¹; HRMS-CI (M/Z): [M+NH ] Calcd. For C22H31N2O10466.1713; found 466.1709. Intermediate 27: (N-(3,4,5-Trimethoxybenzoyl)-2,3,4-tri-O-acetyl-/^D- glucopyranosylamine)uronic acid, methyl ester

3,4,5-Trimethoxy benzoyl chloride (0.76 g, 3.3 mmol), prepared by method D, intermediate 5 (0.59 g, 1.6 mmol) and triphenylphosphine (0.72 g, 2.4 mmol) were treated as described above to afford the title compoimd (0.62 g, 96%); [αp°_D -12.34° (c 1.224, CHC1₃); *H NMR (CDCI3, 300MHz) δ 7.37-7.03 (ms, 3H, 2H aromatic, NH), 5.49 (m, 2H, H-l, 3), 5.14 (m, 2H, H-2, 4), 4.25 (d, IH, fa 10.1, H-5), 3.92, 3.91, 3.89, 3.72 (4s, each 3H, 4xOMe), 2.06, 2.05 (2 signals) (3s, each 3H, 3xOAc); ¹³C NMR (CDCI3, 270MHz) δ 171.1, 169.6, 169.4, 167.1, 166.9 (each s, each OO), 152.9, 152.7, 127.6, 124.3 (4C, each s, each aromatic C), 107.0, 106.9 (2C, each d, aromatic), 78.5 (d, C-l), 73.5, 71.7, 70.4, 69.5 (each d, C-2-5), 60.7, 60.6, 56.1, 52.7 (4C, each q, 4xOMe), 20.6, 20.4, 20.3 (3xOAc, each q); IR (liquid film): υ 3346 (NH), 2950, 2642, 2152, 1742 (CO), 1662 (OO), 1501, 1223, 859, 766 cm-¹; HRMS-CI (M/Z): [M+NH ] . Calcd. For C23H33N₂Oi3 528.1717; found 528.1715.

Intermediate 31 (N-(4-oxo-4-phenyl-butyryl)-2,3,4-tri-O-acetyl-β-D- glucopyranosylamine)uronic acid, methyl ester

Treatment of 3-Benzoylpropionic acid (0.27 g, 1.5 mmol) and intermediate

(0.5 g, 1.5 mmol) as described in method F gave (N-(4-oxo-4-phenyl-butyryl)-

2,3,4-tri-0-acetyl-β-D-glucopyranosylamine)uronic acid, methyl ester (0.2, 35 %): m.p. = 95-97 °C; [α]_D= +2.6° (c 0.01, CHCI3). NMR δ (300 MHz, CDC ) 7.96-7.28 (ms, 5H,aromatic-H), 6.81 (d, IH, J 10, NH), 5.38-5.31 (overlapping signals, 2H, H-l, H-3), 5.15 (apt t, IH, J 9.5, H-4), 5.03 (apt t, IH, J 9.5, H-2), 4.17 (d, IH, J 10, H-5), 3.72 (s, 3H, OCH₃), 3.47 (m, IH, CH(H)), 3.24-3.15 (m, IH, CH(H)), 2.65-2.53 (m, 2H, CH₂); ¹³C NMR (300 MHz, CDCI₃) 198.0, 172.8, 171.3, 69.9, 169.7, 167.5 (each s, each OO), 136.7 (s, aromatic C), 133.5, 128.8, 128.3, 128.3 (each d, each aromatic CH), 78.3, (d, C-l), 74.2, 72.3, 70.4, 69.9 (each d, C-2-5), 53.1 (q, OCH3), 33.6, 30.4 (each t, CH₂), 20.9, 20.8, 20.6 (each q, OAc); IR (KBr) 3355, 2932, 2366, 1753, 1681, 1538, 1449, 1372, 1228, 1099, 1037, 893, 667, 522 cm-¹.

Intermediate 28: N,N'-Di-(tetra-O-acetyl-D-galactopyranosyl)- terephthalamide

R/0.54 (EtOAc: petroleum ether, 3:1); [ ]_D +50.0° (c 0.02, CHCI3); -NMR: δ (300 MHz, CDCb, 1:4 mixture of aβ.ββ anomers) 7.85 (m, 4H, aromatic H), 7.49 (d, IH, /NH,H-I 8.0, NH, α^anomer), 7.23 (d, IH, JNH,HI 9.0, NH, ββ anomer), 6.17 (br signal, IH, H-l, α ?-anomer), 5.20-5.54 (overlapping signals, H-l, ββaxϊoiτxex, H-2— ), 4.09-4.17 (overlapping signals, H-5, H-6a, H-6b), 2.04-2.20 (overlapping signals, OAc); ¹³C-NMR: δ (CDCb) 172.1, 170.6, 170.2, 170.0 (each s, each OO, ββarxomeτ), 171.4, 171.0, 170.8, 170.4 (each s, each OO, cζ -anomer), 166.5 (s, aromatic OO, aβasxoτrxer), 166.3

(s, aromatic OO, /? ?-anomer), 137.3 (s, aromatic C, α 2-anomer), 136.5 (s, aromatic C, ββarxomer), 128.1, 127.9 (2 signals), 127.8 (each d, aromatic C), 79.5, 72.7, 70.9, 68.9, 67.5 (each d, ββanomeτ), 69.0, 67.8, 67.7, 66.6 (each d, αβεnxomer), 61.8 (t, α -anomer), 61.4 (t, ^anomer), 21.0, 20.9, 20.8 (2 signals) (each q, each OAc); υ_maχ (KBr) 2972, 2933, 2356, 2336, 1752, 1673, 1547, 1501, 1368, 1229 cm'¹. HRMS-FAB: found 847.2385 [M+Na]⁺, required 847.2398.

Intermediate 29. Thiophene-2-carboxylic acid-N-(2,3,4,6-tetra-0-acetyl-y#- D-glucopyranosyl)-amide. R/ 0.22 (EtOAc); [ ]_D +15.0° (c 0.02 CHCb); Η-NMR: δ (300 MHz, CDCb)

7.55 (dd, IH, / 1.1, / 5.0, thiophene H), 7.51 (dd, IH, / 1.1, / 3.7, thiophene H), 7.09 (dd, IH, / 3.8, / 4.9, thiophene H), 7.01 (d, IH, J a-i 9.0, NH), 5.34 (apt t, IH, fa = JH-I,NH = 9.0, H-l), 5.38 (apt t, fa = fa = 9.4, H-3), 5.08 (2 x overlapping apt t, 2H, H-2, H-4), 4.34 (dd, IH, J_6a,₆b 12.5, /_6a,5 4.4 , H-6a), 4.11 (dd, IH, J₆b,6_a 12.7, /₆b,5 2.3 , H-6b), 3.90 (ddd, IH, J₅,6a 4.4, J₅,₆b 2.3, fa 10.0, H-

5), 2.08, 2.05, 2.04 (2 signals) (each s, each 3H, each OAc) ; ¹³C-NMR: δ (CDCb) 171.6, 170.6, 169.8, 169.6 (each s, each OO), 161.7 (s, thiophene OO), 137.5 (s, thiophene C), 131.7, 129.2, 127.9 (each d, thiophene C), 79.0, 73.7, 72.6, 70.8, 68.3 (each d), 61.7 (t), 20.7 (3 signals), 20.6 (each q, each OAc); υm_ax (KBr) 3331, 2928, 2851, 1753, 1626, 1753, 1626, 1576, 1536, 1435, 1368,

1244 cm-¹; HRMS-CI: Found 458.1121 [M+H]⁺, required 458.1119.

Intermediate 30 N-(2,3,4,6-tetra-O-acetyl-β-D-mannopyranosyl)-benzamide

R/ 0.53 (EtOAc); [ ]_D +1-67° (c 0.2, CHCb); mp 50-54 °C; -NMR: δ (300 MHz, CDCb) 7.72 (d, 2H, / 8.2, aromatic H), 7.38-7.55 (m, 3H, aromatic H),

7.07 (d, IH, /NH,H-_I 9.0, NH), 5.78 (d, IH, fa, 9.0, H-l), 5.19-5.47 (overlapping signals, H-2-4), 4.32 (dd, IH, /_6a,5 4.9, J_6a,6b 12.4, H-6a), 4.10 (dd, 2H, /₆b,6a 7.3, Jβ_bjs 1.1 H-6b), 3.87 (m, IH, H-5), 2.07 (2 signals), 2.06, 2.05 (each s, each 3H, each OAc); ³C-NMR: δ (CDCb) 170.9, 170.8, 170.0, 169.9, 166.9 (each s, each OO), 133.3 (s, aromatic C), 132.5, 128.9, 127.5 (each d, each aromatic C), 76.9,

74.5, 71.8, 70.7, 65.6 (each d), 62.5 (t), 21.1, 20.9 (2 signals), 20.7 (each q, each OAc); υma (KBr) 3331, 2966, 2935, 2258, 1751, 1667, 1528, 1434, 1370, 1104, 1054 cm-¹. HRMS-CI: found 452.1557 [M+H]⁺, required 452.1559.

Biological assays

Synthesis ofhepaήn-albumin complex

Heparin (456 mg, 37.5 μmol; Fluka (cat. no. 51536) and BSA (17 mg, 0.25 μmol; Fluka (cat. no. 05470) were dissolved in 2.5 mL of 0.2 M potassium phosphate buffer, pH 8.0. Sodium cyanoborohydride (12.5 mg, 198.9 μmol) was then added and the mixture was incubated for 2 days at 37°C. The mixture was dialysed at room temperature against three changes of deionised water and freeze-dried to yield the crude heparm-alburriin complex as a white solid (91mg).

FGF Binding assay

A stock solution of hepar -albumin (5mg/ml) was made in distilled water and diluted to a final working concentration in a buffer containing 0.1M sodium carbonate and 0.1M sodium bicarbonate and coated onto 96-well assay plates. Novel compounds, heparin-albu in and FGF-2 were added to the wells in a lOOμl volume of distilled water and incubated for 4h at 37°C. Wells were then washed sequentially with PBS/ 0.05% T20 to remove any unbound protein and blot dried after each wash. Goat polyclonal IgG antibody was added lOOμl/well and incubated overnight at 37°C. Wells were washed as before. The amount of bound protein retained in the wells was determined by ELISA using an alkaline phosphatase-conjugated rabbit anti-goat IgG heavy and light chain antibody. The ELISA absorbance readings were read at 405nm. Results were analysed using a non-linear curve fitting programme (GraphPad PRISM)

Commercially available heparin albumin (HA) inhibited the binding of FGF to the plate with an ICSQ of 750 ng/mL; synthetic HA inhibited the binding of FGF to the plate with an IC50 of 0.6 ng/mL. Results obtained with novel compounds are summarised in Table 1 below.

Table 1

^* IC50 and EC50 are denned as the concentration of compound required to give Va I max or E max, respectively.

Fibronectin binding assay

A stock solution of heparm-albumin (5mg/ml) was made in distilled water and diluted to a final working concentration in a buffer containing 0.1M sodium carbonate and 0.1M sodium bicarbonate and coated onto 96-well assay plates. Novel compounds, heparm-albumin and fibronectin (300 ng/mL) were added to the wells in a 100 μl volume of distilled water and incubated for 4h at 37°C. Wells were then washed sequentially with PBS/ 0.05% T20 to remove any unbound protein and blot dried after each wash. Anti-fibronectin antibody (1/5000 dilution) was added lOOμl/well and incubated overnight at 37°C. Wells were washed as before. The amount of bound protein retained in the wells was determined by ELISA using an alkaline phosphatase-conjugated rabbit anti-goat IgG heavy and light chain antibody. The ELISA absorbance readings were read at 405nm. Results were analysed using a non-linear curve fitting programme (GraphPad PRISM). Results are shown in Table 2 below.

Table 2

Endothelial cell assay: BAEC were maintained in RPMI 1640 medium supplemented with 10% heat inactivated FCS, 25 mM glutamine, 75 U/mL penicillin and 75 μg/mL streptomycin. Cells were grown to confluency in 75 cm² tissue culture flasks and maintained at 37°C in a humidified atmosphere containing 95% 0₂ and 5% Cθ2- Subcultures were created by passaging using a trypsin/EDTA (0.125%/0.05%) mixture in phosphate buffered saline (PBS), harvested by centrifugation (4 min at 210xg) and seeded at the appropriate density. The methylthiazol tetrazolium (MTT) assay (adapted from

Mosmann et al. (1983) J. Immunol. Meth. 65: 55-63) was used to assess cell viability. Confluent monolayers of BAEC, grown in 24 well tissue culture plates were treated with test compounds (10 μg/mL) at the indicated concentration for 24 h at 37°C. Following aspiration and washing with PBS, each well was incubated with MTT (0.45 mg/ml) in RPMI 1640 for 3 hr at 37

°C. The overlying solution was then aspirated and the cells solubilised by the addition of 1 mL dimethyl sulphoxide. Absorbance was measured at 600 ran and viability expressed as percentage of control (untreated) wells. Statistical significance of differences between group means was determined by ANOVA followed by a post ANOVA Dunnett's test. Results are shown in Table 3.

Table 3

A series of monosaccharide derivatives were evaluated by the ELISA method described above to determine their ability to compete with heparin for binding to FGF-2. The assay was first standardized. The assay results can vary depending on the source of heparin or heparin albumin that is used for the assay. HA (purchased from Sigma) was found to inhibit binding of FGF to HA (Sigma) attached to the plate with an ICSQ of 750 ng/mL and the maximum inhibition (I_maχ) achieved was 85% (at 10⁶ ng/mL). (Fig lb) Heparan sulfate (Sigma, cat. No. H9902) and heparin (Sigma, cat. No. H3149) were evaluated in competition with HA (Sigma). (Fig la) Heparan sulfate had an Imax of 50% at 50 ng/mL; heparin had an I_max of 10%. HA was also prepared by conjugation of heparin (and albumin as described above; the Imax for HA in the FGF-ELISA was 99% with an ICso of 0.61 ng/mL; the I_maχ for the fraction of heparin used for the synthesis of HA was 97% with an ICso of 0.61 ng/mL. The similarity of the data obtained for both heparin and HA indicates that although the HA conjugate is not well defined however, its behaviour and binding profile is very similar to heparin itself. The average M. W. of heparan sulfate used in the assays was 14,200. The biological data is extrapolated to indicate that a compound that would approach the potency similar to heparan sulfate would show up to 50% binding in the ELISA at concentrations of ~5-50 nM. This data correlates reasonably well with the published Kd of 10"⁹ M for heparin binding to FGF (Moscateli (1987), J. Cell Phys. 131: 123).

Twenty-nine monosaccharide conjugates were evaluated in the FGF binding assay. Twelve (examples 1, 5, 8, 10, 14, 18, 19, 21, 24, 27, 29 and 30) were inhibitors of FGF binding to heparm-albumin in the assay. Example 29 was the most potent inhibitor (60% maximum inhibition; 30% inhibition observed at 3.5 nM) and a number of other compounds showed activity in the nM range. Some were less potent (e.g. example 18) showing activity in the mM range. A number of compounds showed ability to stimulate or enhance the binding of FGF to heparin albumin conjugate (examples 2, 4, 11, 39). The mechanism by which the compounds stimulate binding of FGF-2 to HA is unknown. Heparin can bind a number of FGF molecules in a "beads on a string" fashion and it is possible that the compounds could stabilise such aggregates or bind remotely from the heparin binding site and enhance binding through an allosteric mechanism. Perhaps the inhibitors described herein may compete effectively at the heparin binding sites whereas stimulators of HA binding do not but bind instead at another site.

The binding of compounds to Fibronectin in competition with HA (prepared by conjugation of heparin with albumin) was also evaluated by ELISA. Heparin (Fluka, Cat. No.) had an I a of 83% with an ICso of 229 ng/mL in this assay. Of 11 compounds that were evaluated in the Fibronectin binding assay five (examples 3, 10, 17, 19, 21) were found to show inhibitory activity whereas one (example 38) was a stimulator. The most potent inhibitor of fibronectin binding to heparin albumin was example 21 which had a maximum inhibition of 85%; 42.5% inhibition was observed at 51 μM.

Bovine arterial endothelial cell (BAEC) survival was also investigated as a model for testing the ability of the monosaccharide conjugates to modulate endothelial cell signal transduction pathways. These cells express both the FGF receptor and heparan sulfate proteoglycans, and they release FGF-2. This release of FGF-2 not only drives cell proliferation (important in angiogenesis) but also potently suppresses apoptotic cell death; inhibition of

FGF-2 activity using a neutralising anti-FGF-2 antibody results in increased apoptosis. Heparin was evaluated (10 μg/mL) and after 24 h a 32.5% reduction in the number of viable cells was observed. Of 19 monosaccharide conjugates evaluated in the endothelial cell assay four (examples 14, 18, 19, 30) showed > 14.5% decrease in number of viable cells after 24 h; example 30 showed the largest decrease (42%) which was greater than that observed for heparin. The preliminary results indicate that some of the compounds which show inhibitory activity in the binding assay also inhibit cell survival although this does not appear to be a general phenomenon as example 28 does not appear to be active in this assay.

In some cases inhibitors of binding may promote cell proliferation pathways

(mitogenic activity). Three compounds (examples 5, 7, 25) showed stimulation of growth of the BAEC cells. To further show that monosaccharide compounds have mitogenic activity some of the compounds were evaluated for their effects on FR1C- 11 proliferation. These cells have been transfected with the FGF receptor and require both heparin and FGF for growth and are thus suitable for pharmacological evaluation of compounds that modulate FGF activity. Rapid growth of the cells was observed when they were treated with heparan sulfate (lng/rnL) and FGF. The effects of two compounds were evaluated on proliferation of FRlC-11 cells. These cells normally require both heparin and FGF for growth. The results are shown in Fig. 13. One compound (example 13) showed the ability to rapidly increase the growth rates of these cells and at 3.0 nM concentration was more effective than heparin. Interestingly example 13 did not show activity in the ELISA assays but was very effective as a mitogenic agent. Example 19, which was an inhibitor in the FGF- ELISA and was an inhibitor in the endothelial cell assay, was not mitogenic towards the FRIC-11 cells.

In order to establish whether the compounds may have cytotoxic properties, a selection of the compounds (10 μg/mL) were evaluated for their toxicity towards mouse mammary epithelial cells (NMuMG) which was assessed using the methyl tetrazolium (MTT) assay. Heparin reduced the number of viable cells by 9% in these assays. The only compounds to display any activity in this assay were example 8, 10, 11, 30 (7-14%). Examples 6, 7, 13, 14, 17, 18, 19, 22, 24, 25, 27, 28, 29 were inactive towards the epithelial cell line indicating that they are not cytotoxic and that their mechanism is through inhibition of signal transduction pathways or promotion of apoptopic pathways or by another mechanism. Cell morphology of the endothelial cells did not change indicating that the compounds do not have potent cytotoxic action.

The compounds may also work through their inhibition of heparin and FGF as indicated by results of example 29. This compound was inactive in endothelial cell assays but yet it is a potent inhibitor of heparin binding to FGF. It is possible that the compounds (e.g. example 30) also act as very specific glycosidase or glycosyltransferase inhibitors or inhibitors of glucose transport or of glucose metabolism or other mechanism. The stable amide linkage (X containing NHCO for example) found in many of the compounds would confer stability to glycosidases in vivo. In this regard the compounds will have uses in treatments for diabetes, cancer, antibacterial and antiviral infection. It is known for example that the naturally occurring alkaloid, castanospermine is an inhibitor of - and β-glucosidases and that this compound can inhibit angiogenesis by altering endothelial cell glycosylation.

Novel compounds were identified in the present invention that show binding activity to heparin binding proteins. Inhibitors as well as stimulators of FGF binding to heparin and fibronectin binding to heparin were identified. Inhibitors and stimulators of endothelial cell survival pathways were identified. These compounds showed activity in the endothelial cell assays through their ability to inhibit interactions of FGF and heparin. The compounds in general were not very toxic towards epithelial cell lines indicating they may not be cytotoxic. The compounds may also work through another mechanism; they may be acting as inhibitors of glycosidases, glycosyltransferases or enzymes involved in glucose metabolism or by inhibiting glucose transport. The invention alos porvides compounds capable of promoting mitogenesis of cell lines expressing the FGF receptor.

The invention is not limited to the embodiments hereinbefore incorporated by way of example which may be varied in detail.

Claims

Claims

A compound of the Formula

wherein

Ai to A₃ is any one or more of the same or different of

OH; F; or NH_2;

wherein when

B is CO₂H,

X is NR²CO; NR²COCH₂; NR²COCH₂O; NR²COCH=CH;

NR²COCH₂CH₂; NR²COCH₂CH₂CO; NR²S0₂CH₂,

NR²SO₂CH₂O; NR²SO₂CH=CH; NR²SO₂CH₂CH_2; NR²SO₂CH₂CH₂CO; CONR²; CONR²CH₂; CONR²CH₂O;

CONR²CH=CH; CONR²CH₂CH₂; CONR²CH₂CH₂CO; S0₂NR²CH₂; SO₂NR²CH₂O; S0₂NR²CH=CH;

S0₂NR²CH₂CH₂; or SO₂NR²CH₂CH₂CO;

wherein R² is H or alkyl,

and R is benzene; pyridine; pyrazine; thiophene; furan; cyclopropyl; indole; quinoline; naphthalene; chrom-4-enone;

*•< or tetrahydrofuran,

which may be unsubstituted or substituted with any one or more or different of halogen; trifluoromethyl; OMe; Me; NO₂; phenyl; CONH-sugar or NHCO-sugar,

or

X is O; S; S0₂; OCH₂CH₂; SCH₂CH₂; SO₂CH₂CH₂; OCH₂; SCH₂ or SO₂CH₂, and

R is

which may be unsubstituted or substituted with any one or more or different of halogen; OMe; Me; N0₂; trifluoromethyl or OH,

or when

B is CH₂OH

X is NR²CO; NR²COCH₂; NR²COCH₂O; NR²COC₆H4 NR²COCH=CH; NR²COCH₂CH₂CO; NR²SO₂CH₂

NR SO₂CH₂0; NR²SO₂CH=CH; NR²S0₂CH₂CH₂CO CONR²; CONR²CH₂; CONR²CH₂0; CONR²CH=CH; CONR²CH₂CH₂CO; SO₂NR²CH₂; SO₂NR²CH₂O;

SO₂NR²CH=CH; or SO₂NR²CH₂CH₂CO;

wherein R² is H or alkyl,

and R is difluorobenzene;, dichlorobenzene; chlorofluorobenzene; dimethylbenzene; trifluoromethylbenzene; trimethoxybenzene; phenylbenzene; pyrazine; thiophene; furan; cyclopropyl; indole; quinoline; CONH-sugar; NHCO-sugar or

- O

or

R is pyridine, pyrazine, thiophene, furan, cyclopropyl, indole and quinoline containing at least one ring substituent selected from any one or more or different of halogen; trifluoromethyl; OMe; Me; NO₂; or phenyl,

and epimers and pharmacologically acceptable salts, esters, amides and prodrugs thereof.

A compound of the Formula

A₃-^Λ_^O A₂- - -^- _f

A,

wherein

Ai to A₃ is any one or more of the same or different of OH; F or NH_2; wherein when

B is CO₂H

X is NR²CO; NR²COCH₂, NR²COCH₂O; NR²COCH=CH

NR²COCH₂CH₂; NR²COCH₂CH₂CO; NR²S0₂CH₂ NR²SO₂CH₂0; NR²SO₂CH=CH; NR²SO₂CH₂CH_2i

NR²SO₂CH₂CH₂CO; CONR²; CONR²CH₂; CONR²CH₂0 CONR²CH=CH; CONR²CH₂CH₂; CONR²CH₂CH₂CO SO₂NR²CH₂; SO₂NR²CH₂O; S0₂NR²CH=CH:

SO₂NR²CH₂CH₂ or SO₂NR²CH₂CH₂CO,

wherein R² is H or alkyl,

R is benzene; pyridine; pyrazine; thiophene; furan; cyclopropyl; indole; quinoline; naphthalene; chrom-4-enone;

or tetrahydrofuran,

which may be unsubstituted or substituted with any one or more or different of halogen; trifluoromethyl; OMe; Me; N0₂; phenyl; CONH-sugar or NHCO-sugar,

and epimers and pharmacologically acceptable salts, esters, amides and prodrugs thereof.

3. A compound of the Formula

wherein

Ai to A₃ is any one or more of the same or different of OH; F or NH₂.

wherein

B is CO₂H

X is O, S, SO₂; OCH₂CH₂;, SCH₂CH₂; SO₂CH₂CH₂;, OCH₂; SCH₂ or SO₂CH₂,

and

which may be unsubstituted or substituted with any one or more or different of halogen; OMe; Me; NO₂ or OH

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof. A compound of the Formula

wherein

Ai to A₃ is any one or more of the same or different of OH; F or NH_2;

B is CH₂OH

X is NR²CO; NR²COCH₂; NR²COCH₂O; NR²COCH=CH; NR²COCH₂CH₂CO; NR²SO₂CH₂; NR²SO₂CH₂O,- NR²SO₂CH=CH; NR²SO₂CH₂CH₂CO; CONR²; CONR²CH₂; CONR²CH₂O; CONR CH=CH; CONR²CH₂CH₂CO; SO₂NR CH₂; SO₂NR²CH₂0; SO₂NR²CH=CH or SO₂NR²CH₂CH₂CO;

wherein R² is H or alkyl

and R is difluorobenzene; dichlorobenzene; chlorofluorobenzene; dimethylbenzene; trifluoromethylbenzene; trimethoxybenzene; phenylbenzene; pyrazine; thiophene; furan; cyclopropyl; indole; quinoline; CONH-sugar; NHCO-sugar; or

-\ J

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof. compound of the formula

wherein

Ai to A₃ is any one or more of the same or different of OH; F or NH_2;

B is CH₂OH

X is NR²CO; NR²COCH₂; NR²COCH₂0; NR²COCH=CH

NR²COCH₂CH₂CO; NR²SO₂CH₂; NR²SO₂CH₂O; NR²SO₂CH=CH NR²SO₂CH₂CH₂CO; CONR²; CONR²CH₂; CONR²CH₂O CONR²CH=CH; CONR²CH₂CH₂CO; SO₂NR²CH₂; S0₂NR²CH₂O SO₂NR²CH=CH or S0₂NR²CH₂CH₂CO;

wherein R² is H or alkyl,

or

R is pyridine, pyrazine, thiophene, furan, cyclopropyl, indole and quinoline which containing at least one ring substituent selected from any one or more or different of halogen; trifluoromethyl; OMe; Me; NO₂ or phenyl,

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

6. A compound of the formula

wherein

B is CH₂OH

X is NR²CO; NR²COCH₂; NR²COCH₂O; NR²COCH=CH NR²COCH₂CH₂CO; NR²SO₂CH₂; NR²SO₂CH₂O; NR²SO₂CH=CH

NR²SO₂CH₂CH₂CO; CONR²; CONR²CH₂; CONR²CH₂0 CONR²CH=CH; CONR²CH₂CH₂CO; SO₂NR²CH₂; SO₂NR²CH₂0 SO₂NR²CH=CH; or SO₂NR²CH₂CH₂CO,

and

R is thiophene, unsubstituted or substituted with one or more or different of halogen; trifluoromethyl; OMe; Me; NO₂; phenyl; CONH-sugar or CONH-sugar,

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

7. A compound as claimed in any of claims 1 to 6 wherein the halogen is selected from any one or more of F, Cl, Br or I.

A compound as claimed in any of claims 1 to 7 wherein the sugar is selected from any one or more of glucose, galactose, mannose, glucuronic acid or iduronic acid.

9. A compound as claimed in any of claims 1 to 8 wherein the R group is mono, di or tri-substituted.

10. A compound of the Formula

wherein

Ai to A₃ is any one or more of the same or different of

H; OH; F or NHAc,

X is NHCO or NHCOCH₂O,

and

R¹ is an aromatic or heteroaromatic group, which may be unsubstituted or substituted or a group selected from any one or more of

XA

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

11. A compound as claimed in claim 10 wherein Ai to A are each hydroxy.

12. A compound as claimed in claim 10 or 11 wherein the aromatic or heteroaromatic group is selected from any one of benzene; pyridine; thiophene; pyrazine or indole.

13. A compound as claimed in any of claims 10 to 12 wherein the aromatic or heteroaromatic group is substituted with any one or more or different of halogenjtrifluoromethyl; OMe; Me; NO₂; phenyl; CONH-sugar or NHCO- sugar.

14. A compound as claimed in any of claims 10 to 13 wherein the aromatic or heteroaromatic group is substituted with fluorine or chlorine.

15. A compound of the Formula

wherein

Ai to A₃ is any one or more of the same or different of

H; OH; F or NHAc;

X is NHCO; OCH₂CH₂; NHCOCH₂; NHCOCH₂CH₂CO or NHCOCH₂O

and

R is selected from any one or more of

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

16. A compound as claimed in claim 15 wherein Ai to A₃ are each hydroxy.

17. A compound of the Formula

wherein

X is O or OCH₂CH₂,

and

R is selected from any one or more of

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

18. A compound of the Formula

wherein

B is CO₂H or CH₂OH

X is NHCOCHzO or NHCO,

and R is selected from any one or more of

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

19. A compound of formula

in

B is CO₂H;

X is OCι-C₆alkyl; and

R is a cycloalkanone,

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

20. A compound of formula

wnerein

A'ι to A₃ is OH; B is CO₂H;

X is NR³CO; NR³COC_]-6alkenyl; NR³COC_]-6 alkyl; NR³CO; NR³COCH₂0;

wherein R³ is H, alkyl, alkenyl or alkynyl, and R is H, aromatic group or cycloalkyl group which may be substituted or unsubstituted,

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

21. A compound as claimed in claim 1 of the Formula

wherein

Ai to A₃ is any one or more of the same or different of

OH or F

X is NHCO or NHCOCH₂O,

and

Ris

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

2. A compound as claimed in claim 1 of the Formula

wherein

Ai to A₃ is any one or more of the same or different of OH or F;

X is NHCO; NHCOCH₂; NHCOCH₂CH₂CO or NHCOCH₂O

and Ris

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

23. A compound as claimed in claim 1 of the Formula

wherein

X is O or OCH₂CH₂, or

and Ris

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

24. A compound as claimed in claim 1 of the Formula

wherein

B is CO₂H or CH₂OH

X is NHCOCH₂0 or NHCO,

and Ris

and epimers, pharmacologically acceptable salts, esters, amides and prodrugs thereof.

25. A compound selected from the following

(N-(Benzoyl)-2,3,4-tri-O-acetyl->9-D-glucopyranosylamine)uronic acid, methyl ester;

Succinimidoethyl-2,3,4-tri-0-acetyl-β-D-glucopyranuronic acid, methyl ester;

Phthalimidoethyl-2,3,4-tri-0-acetyl-β-D-glucopyranuronic acid, methyl ester;

(l_>4-Dioxaspirol[4,5]dec-2-yl)-2,3,4-tri-0-acetyl-β-D-glucopyranuronic acid, methyl ester; (3-Benzoylphenyl)-2,3,4-tri-O-acetyl-β-D-glucopyranuronic acid, methyl ester;

N-(2-Biphenylcarbonyl)-2,3,4-tri-0-acetyl-/?-D-glucopyranosylamine) uronic acid, methyl ester;

(N-(2-Phenylquinoline-4-carbonyl)-2,3,4-tri-O-acetyl-5-D-glucopyranosyl amine) uronic acid, methyl ester; (N-(4-Chlorophenylacetyl)-2,3,4-tri-0-acetyl-β-D-glucopyranosylamine) uronic acid, methyl ester;

(N-(3-methyl-2-but-2-enoly)-2,3,4-tri-O-acetyl-β-D-glucopyranosylamine) uronic acid, methyl ester; (N-(3-furan-2-yl-acryloyl)-2,3,4-tri-0-acetyl-β-D-glucopyranosylamine) uronic acid, methyl ester;

(N-(4-Biphenylaceιyl)-2,3,4-tri-0-acetyl-β-D-glucopyranosylamine)uronic acid, methyl ester; (N-(2-Methylpentanoyl)-2,3,4-tri-O-aceryl-β-D-glucopyranosylamine) uronic acid, methyl ester;

(N-2-cyclopropanecarbonyl)-2,3,4-tri-0-acetyl-β-D-glucopyranosylamine) uronic acid, methyl ester;

(N-(2,4-dichloroρhenoxyacetyl)-2,3,4-tri-O-acetyl-β-D-glucopyranosyl- amine)uronic acid, methyl ester;

(N-(2-Pyrazinoyl)-2,3,4-tri-O-acetyl-y5-D-glucopyranosylamine)uronic acid, methyl ester;

(N-(2-Thiophenoyl)-2,3,4-tri-O-acetyl-_/c D-glucopyranosylamine)uronic acid, methyl ester; (N-(2-Pyridine-4-carbonyl)-2,3,4-tri-O-acetyl-&-D-glucopyranosylamine) uronic acid, methyl ester;

(N-(2-Chloro-4-nitrobenzoyl)-2,3,4-tri-O-acetyl-_>^-D-glucopyranosyl- amine)uronic acid, methyl ester;

(N-(3,4-Difluorobenzoyl)-2,3,4-tri-0-acetyl- ?-D-glucopyranosylamine) uronic acid, methyl ester;

(N-(3-Trifluoromethylbenzoyl)-2,3,4-tri-O-acetyl-^-D-glucopyranosyl-amine) uronic acid methyl ester;

(N-(3,5-Dimethylbenzoyl)-2,3,4-tri-0-acetyl-_/t?-D-glucopyranosylamine) uronic acid, methyl ester; (N-(3_J4,5-Trimethoxybenzoyl)-2,3,4-tri-O-acetyl-_/5-D-glucopyranosyl- amine)uronic acid, methyl ester;

[N-(naphthalene-2-carbonyl)-2,3,4-tri-0-acetyl- 5-D-glucopyranosylamine] uronic acid, methyl ester; [N-(2-Thiophenoyl)-2,3-di-0-acetyl-4,5-anhydro- 5-D-glucopyranosyl-amine] uronic acid, methyl ester;

[N- (Benzoyl) -2,3-di-O-acetyl-4,5-anhydro-_>-?-D-glucopyranosylamine] uronic acid, methyl ester; [N- (3,5-Dimethyιbenzoyl) -2,3-di-0-acetyl-4,5-anhydro- ?-D-glucopyran- osylamine] uronic acid, methyl ester

26. A compound selected from the following

Succinimidoethyl-β-D-glucopyranuronic acid;

Phthalimidoethyl-β-D-glucopyranuronic acid;

[l,4-Dioxaspirol[4,5]dec-2-yl]-β-D-glucopyranuronic acid;

3-Benzoylphenyl-β-D-glucopyranuronic acid;

(Ν- (4-Chlorophenylacetyl) -β-D-glucopyranosylamine)uronic acid; (Ν-(3-methyl-2-but-2-enoly)-β-D-glucopyranosylamine)uronic acid;

(N- (3-Furan-2-ylacryloyl) -β-D-glucopyranosylamine) uronic acid;

(N-(2-Methylpentanoyl)-β-D-glucopyranosylamine)uronic acid;

((N-cyclopropanecarbonyl) -β-D-glucopyranosylamine)uronic acid;

(N-((2,4-Dichlorophenoxy)acetyl)-β-D-glucopyranosylamine)uronic acid; (N-(Benzoyl)- ^D-glucopyransylamine)uronic acid;

(N-(3-Trifluoromethylbenzoyl)-ff-D-glucopyranosylamine)uronic acid;

(N- (3,5-Dimethylbenzoyl) - ?-D-glucopyranosylamine)uronic acid;

(N-(3,4,5-Trimethoxybenzoyl)-y-?-D-glucopyranosylamine)uronic acid;

(N-(Biphenyl-2-carbonyl)- ?-D-glucopyranosylamine)uronic acid; (N-(2-Phenylquinoline-4-carbonyl)-/?-D-glucopyranosylamine)uronic acid;

(N-(2-Pyrazinoyl)->5-D-glucopyranosylamine)uronic acid;

(N-(2-Thiophenoyl)-5-D-glucopyranosylamine)uronic acid;

(N-(2-Pyridine-4-carbonyl)-5-D-glucopyranosylamine) uronic acid; (N-(2-Chloro-4-nitrobenzoyl)- WD-glucopyranosylamine)uronic acid;

[N-(3,4-Difluorobenzoyl)-/?-D-glucopyranosylamine] uronic acid;

[N-(naphthalene-2-carbonyl)- 0-D-glucopyranosylamine] uronic acid;

[N-(lH- dole-2-carbonyl)- ?-D-glucopyranosylamine] uronic acid; [N- (3,5-Dimethylbenzoyl) -anhydro-5-D-glucopyranosylamine]uronic acid;

(Ν-(4-Oxo-4-phenylbutyryl)-β-D-glucopyranosylamine)uronic acid;

(N-3(lH-indol-3-yl)-propionyl)-β-D-glucopyranosylamine)uronic acid;

(N-(4-biphenylacetyl-2,3,4-tri-O-acetyl-β-D-glucopyranosylamine)uronic acid;

(N-3-methyl-4-oxo-2-phenyl-4H-chromene-8-carbonyl-β-D-glucopyran- osylamine) uronic acid;

(N- (4-Oxo-4-phenyl-butyryl) -β-D-glucopyranosylamine)uronic acid

N,N ' -Di- (β-D-galactopyranosyl) -terephthalamide

Thiophene-2-carboxylic acid-N- (β-D -glucopyranosyl) -amide

[N-(3,4-Difluorobenzoyl)- ?-D-glucopyranosylanιine] uronic acid [N-(naphthalene-2-carbonyl)-ff-D-glucopyranosylamine] uronic acid

[N-(lH-indole-2-carbonyl)- ?-D-glucopyranosylamine] uronic acid

(Ν-3 (lΗ-indol-3-yl) -propionyl) -β-D-glucopyranosylamine)uronic acid

(N-(4-biphenylacetyl-2,3,4-tri-O-acetyl-β-D-glucopyranosylamine) uronic acid

N-(3-Methyl-4-oxo-2-phenyl-4H-chromene-8-carbonyl)-β-D- glucopyranosylamine)uronic acid

N,N'-Di(β-D-glucopyranuronosyl)-terephthalamide

(N-(Tetrahydrofuran-2-carbonyl)--?-D-glucopyranosylamine)uronic acid

27. A pharmaceutical composition comprising a compound as claimed in any of claims 1 to 26 including a pharmaceutically acceptable carrier or diluent.

28. Use of a compound as claimed in any of claims 1 to 26 for the preparation of a medicament for the treatment and/or prophylaxis of atherosclerosis and human coronary heart disease, chronic myocardial ischemia, rheumatoid arthritis; ulcerative colitis, inflammatory bowel disease, crescentic glomerulonephritis, diabetic retinopathy; retinal ischemia, glomemlosclerosis, age-related macular degeneration; psoriasis, intermittent claudication, bacterial meningitis.

29. Use of a compound as claimed in any of claims 1 to 26 for the preparation of a medicament for the stimulation of bone formation in osteopenic disorders; stimulation of muscle, nerve, cornea (retinal) and colonic and other tissue repair; disease associated with nerve tissue regeneration, such as spinal cord injury, multiple sclerosis, Alzheimers disease, Parkinson's disease and for healing of gastric, duodenal, colonic and leg ulcers.

30. Use of a compound as claimed in any of claims 1 to 26 in the preparation of a medicament for use in the modulation of fibronectins.

31. Use of a compound as claimed in any of claims 1 to 26 in the preparation of a medicament for use in the modulation of fibroblast growth factors.

32. Use of a compound as claimed in any of claims 1 to 26 in the preparation of a medicament for use in the modulation of endothelial cell survival or proliferation.

33. Use of a compound as claimed in any of claims 1 to 26 in the preparation of a medicament for use in the modulation of glycosaminoglycan binding proteins.

34. Use of a compound as claimed in any of claims 1 to 26 in the preparation of a medicament for use in the modulation of heparin binding proteins.

35. Use of a compound as claimed in any of claims 1 to 26 in the preparation of a medicament for use in the inhibition of glycosidases or glycosyltransferases or saccharide transport or metabolism in cells.

36. Use of a compound as claimed in any of claims 1 to 26 in the preparation of a medicament for the treatment and/or prophylaxis of cancer.

37. Use as claimed in claim 36 wherein the cancer is invasive breast cancer, pancreatic cancer, progressive multifocal leukoencephalopathy, Kaposis- sarcoma, prostrate cancer, testicular cancer, endocrine related cancers, ovarian cancer, neuroblastoma, human-malignant mesothelioma, renal cell carcinoma, leukemia, gastric carcinoma, fibromatosis, lung cancer, carcinoma of the bladder, non-Hodgkin's lymphoma, colo-rectal cancer; benign prostatic hyperplasia, venous neointimal hyperplasia, intimal hyperplasia.

38. Use of a compound of the formula

wherein

X is NHCOCHzO or NHCO,

and

R is benzene,

in the preparation of a medicament for use in the modulation of fibroblast growth factors.