AU730910B2 - Protected aminosugars - Google Patents

Description

WO 98/38197 PCT/AU98/00131 PROTECTED AMINOSUGARS This invention relates to methods for synthesis of oligosaccharides, especially those oligosaccharides which comprise amino sugar residues. In particular the invention relates to methods for solution phase, solid phase or combinatorial synthesis of oligosaccharides.

BACKGROUND OF THE INVENTION Aminosugars are important constituents of various glycoconjugates (Schmidt and Kinzy, 1994). Examples include peptidoglycans, mucopolysaccharides, glycopeptides and proteins, oligosaccharides of human milk, and blood group determinants. They are often also encountered in bacterial and tumour-associated carbohydrate antigens, predominantly in the N-acetylated form or N-acylated with an aspartic acid residue (Toyokuni and Singhal, 1995). It is therefore evident that these biological glycoconjugates are of immense interest to the medicinal chemist, and therefore that there is a great need in the art to be able to synthesise these compounds in a facile and costeffective manner.

Oligosaccharide synthesis using aminosugars requires the presence of a suitable amino protecting group.

A number of protecting groups have been proposed, but so far all of the agents which are available suffer from serious disadvantages. For example, glycosylation with donors derived from 2-N-acetyl protected aminosugars proceeds via neighbouring group participation; however, formation of the relatively stable oxazoline intermediate dramatically reduces the overall speed and yield of the reaction (Zurabyan et al, 1994). Therefore, various 2 -deoxy-2-aminosugar donors, displaying the neighbouring group activity described, but lacking the ability to form stable oxazolines, have been developed; the most widely used of these are the phthalimido protected monomers (Sasaki et al, 1978). The phthalimide group participates WO 98138197 PCT/AU98/00131 2 strongly during glycoside formation and gives excellent stereocontrol of the 1,2-trans-glycoside product (Lemieux et al, 1982), furthermore the aminosugar donors do not form stable orthoamides (Lemieux et al, 1982) and cannot form oxazolines. The major disadvantage of using the phthalimide group lies in the vigorous conditions required for its removal, namely heating with methanolic hydrazine, which often results in partial product decomposition.

Strongly basic conditions are also required for the removal of the N-sulfonyl (Griffith and Danishefsky, 1990) and Nhaloacetyl protecting groups (Shapiro et al, 1967), resulting in similar problems.

The allyloxycarbonyl (Alloc) protected amino sugar donors display a similar activity to their phthalimide counterparts when employed under Lewis acidcatalysed conditions. However, the Alloc group has the advantage that it can be removed under extremely mild conditions, using tetrakis (triphenylphosphine) palladium in the presence of a mild base (Hayakawa et al, 1986). The major disadvantage associated with the Alloc group lies in its ability to form a stable oxazolidinone intermediate, which in the presence of unreactive acceptors tends to remain as the major product, and reduces the speed and yield of the reaction (Boullanger et al, 1987). 2,2,2- Trichloroethyl-protected aminosugars contain a strongly participating group that, unlike phthalimide, does not deactivate adjacent hydroxyl groups which may subsequently be required as glycosyl acceptors. They can be removed under relatively mild and selective conditions, using zinc and acetic acid, and do not form oxazoline intermediates during glycosylation. However, this protecting group has the disadvantage that benzyl groups cannot be introduced without premature loss of the protecting group as well (Imoto et al, 1987).

Tetrachlorophthaloyl-protected aminosugar donors have been demonstrated to afford high yields of 1,2-transglycosides (Castro-Palomino and Schmidt, 1995), even in the WO 98/38197 PCT/AU98/00131 3 presence of poorly reactive acceptors. Once more, however, the NaBH4-mediated deprotection is the limiting factor for this particular protecting group.

The azide group has received much attention in aminosugar chemistry, since it serves as a masked, nonparticipating amino functionality, thereby allowing the synthesis of 1,2-cis-linked 2-amino-2-deoxy glycosides (Palsen, 1982). However the preparation of 2-azido-2-deoxy sugars is protracted, costly, and often dangerous, using either azidonitration (Lemieux and Ratcliffe, 1979), diazotransfer reactions (Buskas et al, 1994), azidochlorination (Bovin et al, 1986), nitrosation of N-benzyl derivatives (Dasgupta and Garegg, 1989) or reactions of 1,6anhydrosugars (Tailler et al, 1991 and Paulsen and Stenzel, 1978).

Other non-participating protecting groups that have been reported are 2,4-dinitrophenyl (Kaifu and Osawa, 1977) and p-methoxybenzylimino (Mootoo and Fraser-Reid, 1989), both of which are complicated to introduce and require harsh deprotection conditions which result in loss of product.

A hydrazine-labile primary amino-protecting group, N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), has been reported for protection of lysine side chains during SPPS (Bycroft et al, 1993). This group was modified for use as a carboxy-protecting group in SPPS when the 2 -(3-methylbutyryl)dimedone analogue of 2-acetyldimedone was condensed with 4-aminobenzylalcohol to afford 4 4 ,4-dimethyl-2,6-dioxocyclo-hexylidene)-3methylbutyl]-amino}benzyl ester (ODmab) (Chan et al, 1995).

These two protecting groups were reported to be stable to the Fmoc deprotecting conditions widely used in solid phase peptide synthesis (SPPS), ie 20% piperidine in dimethylformamide (DMF).

Dde has been widely used in the field of SPPS as an orthogonal amino protecting group to the well established Fmoc/t-Boc methodology (Fields and Noble, 4 1990). Until now its use has remained within this area, and therefore its use as a protecting group in the field of carbohydrate chemistry is novel. In particular, the use of Dde or ODMab in oligosaccharide synthesis has not been suggested.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

We have now surprisingly found that Dde can be used as a non-participating amino sugar protecting group, 20 which can be introduced and removed in a facile and costeffective manner. We have shown that the vinylogous amide protection afforded by the Dde type group is achieved by simply refluxing the unprotected amino sugar with the precursor, eg. 2-acetyldimedone in the case of Dde, in 25 anhydrous ethanol. Using a Dde-protected aminosugar, we ooooe: S• have performed a variety of chemical modifications upon the cee protected molecule in order to demonstrate the stability of this vinylogous amide type protection towards commonly c:o encountered reactions involved in carbohydrate oooo S: 30 modification.

SUMMARY OF THE INVENTION In one aspect, the invention provides a compound useful as a reagent for solution and/or solid phase synthesis of sugar-containing compounds, comprising a sugar carrying one or more primary amine groups protected with a 4a 2-substituted-1,3-dioxo compound of General Formula I or General Formula II: 0 0 R1 O

NO

2 0 which WO 98/38197 PCT/AU98/00131 5

R

1 and R 2 may be the same or different, and is each hydrogen or C 1 -4 alkyl, R' is an amino sugar, a glycosylamine, or an oligosaccharide comprising at least one aminosugar or one glycosylamine unit, in which the sugar is coupled via an amino group, and R" is alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl or substituted cycloalkyl.

Any sugar or oligosaccharide bearing an amino group may be used.

In a preferred embodiment, the invention provides a reagent for solution phase synthesis of sugar-containing compounds, comprising a cyclic 2-substituted-l,3-dioxo compound of General Formula I or II as defined above, in which R' is as defined above.

The compounds of the invention are suitable for use in methods of solid-phase oligosaccharide synthesis, in which sugar units are covalently linked to a resin. Any suitable linker compound may be used. For example, the covalent linkage to the resin may suitably be provided by a -CONH-, -COO-, -NHCONH-, -NHCSNH, or -NHNH- grouping, eg. Spacer-CONH-resin, Spacer-O-resin, Spacer-S-resin, Spacer-C0 2 -resin, Spacer-CH=N-resin, Spacer-NHCONH-resin, Spacer-NHCSNH-resin, Spacer NHNHresin. Other possible covalent linking groups will be known to those skilled in the art. It is contemplated that linkers and methods described in our International Patent Application No. PCT/AU97/00544 filed on 26 August 1997, are suitable for use with the compounds of this invention. The entire disclosure of PCT/AU97/00544 is incorporated herein by this cross-reference. These linker systems enable solid phase synthesis of oligosaccharides under mild conditions analogous to those used for SPPS.

The resin may be any resin which swells in water and/or in an organic solvent, and which comprises one of the following substituents: halogen, hydroxy, carboxyl, SH,

NH

2 formyl, SO 2

NH

2 or NHNH 2 for example WO 98/38197 PCT/AU98/00131 6 methylbenzhydrylamine (MBHA) resin, amino or carboxy tentagel resins, paraaminomethylbenzyl (PAM) resin, or 4-sulphamylbenzyl AM resin. Other suitable resins will be known to those skilled in the art.

Thus in a second aspect the invention provides a linker-saccharide complex, comprising a linker group and a saccharide compound comprising a protecting group of general formula I or II as defined above, in which the group R' is as defined above.

In a third aspect the invention provides a resinlinker-saccharide support for solid-phase oligosaccharide synthesis, comprising a linker group, a resin, and a starting saccharide compound comprising a protecting group of General Formula I or General Formula II as defined above, in which the group R' is as defined above.

Any suitable linker may be used. Again, it is contemplated that linkers and methods described in PCT/AU97/00544 may be used.

In a fourth aspect the invention provides a method of solid-phase synthesis of oligosaccharides, comprising the step of sequentially linking mono- or oligosaccharide groups, one or more of which is protected as described above, to a resin-linker-saccharide support as described above.

In a fifth aspect the invention provides a method of solution phase synthesis of oligosaccharides, comprising the step of sequentially linking mono- or oligosaccharide groups to a linker-saccharide complex as described above.

These methods are particularly useful for combinatorial synthetic applications. The solid phase or solution phase method of the invention may, for example, be used for combinatorial synthesis of aminoglycoside compounds. It will be appreciated that the sequential linkage may be effected either enzymically or by chemical means.

The invention also provides a kit for solid phase synthesis, solution phase synthesis, or combinatorial WO 98/38197 PCT/AU98/00131 7 synthesis of oligosaccharides, comprising a linkersaccharide complex or a resin-linker-saccharide support according to the invention, as described above. The kit may optionally also comprise one or more further reagents such as partially or differentially activated, fully protected saccharides, protecting agents, deprotecting agents, resins and/or solvents suitable for solid phase or combinatorial synthesis. The person skilled in the art will be aware of suitable further reagents. Different types of kit can then be chosen according to the desired use.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

DETAILED DESCRIPTION OF THE INVENTION Ac Bu Dde

DMF

EtOH

FAB-MS

Me MeOH Nde NHNde

NMR

ODmab

SPPS

TBDMS

tBu Trt Abbreviations used herein are as follows: acetyl butyl N-l-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-ethyl N,N'-Dimethylformamide Ethanol Fast atom bombardment mass spectrometry Methyl Methanol l-(4-Nitro-l, 3 -dioxoindan-2-ylidene) ethyl NH-l-(4-nitro-l,3-dioxoindan-2-ylidene)ethyl Nuclear magnetic resonance 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)- 3-methylbutyl]-amino)benzyl alcohol solid phase peptide synthesis tert-butyl dimethyl silyl tert-butyl trityl WO 98/38197 PCT/AU98/00131 8 The invention will now be described in detail by way of reference only to the following non-limiting examples, in which the structures of individual compounds are as summarised in the following tables.

Table 1

C

U)

m m m Compound R 3 R 4 RS5 R 6 R 7 R8 R 9 R10 RlR 12 1 OH/H OH/H NHDde H H OH OH H CH 2 0H H 2 IH AcO NHDde H H OAc OAc H CH 2 0ac H 3 H Br NHDde H H OAc OAc H CH 2 0ac H 4 H/OMe OMe/H NHDde H H OAc OAc H CH 2 0ac H Isothiouroniumn H NHDde H H OAc OAc H CH 2 0ac H salt 6 SMe H NHDde H H OAc OAc H CH 2 Oac H 7 H OBn NHDde H H OH OH H CH 2 0H H 8 N 3 H NHDde H H OAc Oac H CH 2 0ac H 9 SH H NHDde H H OAc Oac H CH 2 0ac H H OBn NHDde H H OH Benzylidine H Benzylidine H 11 H 103n Nl e H H OAc IOac H -CH 2 Oac H 12 JOH/H jH/OH INHDde IH IH OAc O0ac IH 1CH 2 0aC H Table 1 (continued) Compound R' 4 Rs R6 R 7 R8 R 9 0 R1R 1 13 Imidate/H H/Imidate NHDde H H OAc Qac H CH 2 Oac H 14 H OBn NHDde H H OH OH H CH 2 0trt H H OBn NHDde H H OH OH H CH 2 0TBDMS H 16 NH 2 H NHDde H H OAc Oac H CH 2 0ac H 17 OAc H NHDde H H OAc Oac H CH 2 Odmab H 18 NH 2 H NHDde H H OAc Oac H CH 2 0ac H 19 NHDde H NH-Ac H H 'OAc Oac :H CH 2 0ac H H OBn NHDde H H OH Isopropylidene H Isopropylidene H C 21 H/OH OH/H NHDde H H OH H OH CH 2 0H H 22 IH/OH OH/H NHNde H H OH OH H CH 2 0H H 23 IH OAc NHNde H H IOAc OAc H CH 2 Oac H WO 98/38197 WO 9838197PCT/AU98/00131 11 Table 2 Compound No. R' 1R2 R' R 4 IR 5 R IR 7 24 H H H H NHDde CH 2 OH H Table 3 OcOAC Oc/

ACO

Compound No. R

N

3 26

NH

2 27 jNHDde Example 1 Synthesis of Dde protected aminosugars 2-Deoxy-2- 4-dimethyl 6-dioxo-cyclohex-lylidene) ethyl amnl-D-glucopyranose (1) Sodium (143 mg, 6.21 mmol) was added to abs.

methanol (30 ml) and the reaction mixture was stirred for 5 min. D-glucosamine hydrochloride (1.34 g, 6.21 minol) was added to the resulting clear solution and the reaction WO 98/38197 PCT/AU98/00131 12 mixture was stirred at room temperature for another 5 min.

2- Acetyldimedone (1.69 g, 9.32 mmol) was added and the reaction mixture was stirred under reflux for 5 hours. The reaction mixture was cooled and the product was precipitated'by ether (200 ml) resulting in 2-Deoxy-2-[1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-ethylamino]-Dglucopyranose (1.66 g, 77.9%).

Rf 0.37 (MeCN/H 2 0 10:0.5); FAB MS C 1 6

H

2 5 N0 7 (343.33) m/z 366 (100), 268 246 224 1H NMR (D 2 0) 8 5.12 H-1 3.95-3.25 6H, sugar H), 2.38, 2.36 (2s, 3H, CH 3 2.28, 2.27 (2s, 4H, 2 CH 2 0.85 6H, 2 CH 3 Example 2 Synthesis of Dde-protected O-acylated aminosugars 2-Deoxy-2-[1- 4 -dimethyl-2, 6-dioxocyclohex-1-ylidene)ethylamino]-1,3,4,6-tetra-O-acetyl-a-D-glucopyranose (2) A mixture of 2-deoxy-2-[1-(4,4-dimethyl-2,6dioxocyclohex-1-ylidene)ethylamino]-D-glucopyranose (1.55 g, 4.51 mmol), pyridine (11 ml) and acetic anhydride (20 ml) was stirred at room temperature overnight. The reaction mixture was evaporated, and the product was crystallised from MeOH (10 ml) at -15 0 C to give 2-Deoxy-2- [1-(4,4-dimethyl-2,6- dioxocyclohex-1-ylidene)ethyl-amino]- 1, 3 ,4,6-tetra-O-acetyl-a-D-glucopyranose (1.95 g, 86%).

Rf 0.35 (Hexane/EtOAc 1:1); FAB MS C 2 4

H

3 3 NO11 (511.50) m/z 534 512 [M+H] (100), 452 338 1H NMR (CDC13) 6 13.70 1H, NH), 6.22 1H, H-l,

J

1 2 =3.66 Hz), 5.40 1H, 5.16 1H, H-4), WO 98/39197 PCT/AU98/00131 13 4.36 (dd, 1H, 4.25 (in, 1H, H1-5), 4.13 (dd, 1H1, H-2), 4.05 (dd, 1H, H1-6), 2.58 3H, 0113), 2.35 4H, 2 OH 2 2.09, 2.03, 1.97 (3s, 911, 3 AcO), 1.00 2 OH 3 Example 3 Synthesis of Dde-protected halogenated aminosugars 2-DeoxyV-2-[1- 4-dimethyl 6-dioxocyclohex-1-ylidene) ethylamino] 6 -tri-O-acetyl-a-D-glucopyranosy1 bromide (3) A mixture of 2-Deoxy-2-[1-(4,4-dimethyl-2,6dioxocyclohex-l-ylidene)ethylamino) 6-tetra-O-acetyla-D-glucopyranose (100 mng, 0.19 inmol) and HBr in acetic acid (1.0 ml) was stirred at room temperature for min. The reaction mixture was diluted with cold 0112012 (10 ml), washed twice with cold H 2 0 (30 ml), saturated NaHCO 3 solution (20 mil) and with 1120 again (20 ml). The organic phase was dried over MgSO 4 and evaporated, giving 2-Deoxy-2- 4-dmmethyl-2, 6-dioxocyclohex-1-ylidene) ethylamino] 6 -tri-O-acetyl-a-D-glucopyranosyl bromide (95 mg, 91%).

Rf 0.35 (Hexane/EtOAc 1:1); FAB MS C 22

H

30 BrN0 9 (532.37) m/z 534 (100), 452 (45) 441 (42) 338 (77).

111 NMR (ODC1 3 8 13.83 1H, NH), 6.41 111, H-1,

J

1 2 =3.65 Hz), 5.52 Ct, 1H1, 5.20 1H, 11-4), 4.38 Cm, 2H1, H-61, H1-2), 4.24 (mn, 1H, 4.14 Cdd, 111, 2.62 Cs, 3H, 0113), 2.41 4H, 2 0112), 2.11, 2.04, 1.96 (3s, 9H, 3 AcO), 1.02 6H1, 2 0113).

-WO 98/3&1197 PCT/AU98/00131 14 Example 4 Synthesis of Dde-protected 0-alkylated aminosugars Methyl 2-Deoxy-2-[l- 4-dimethyl-2, 6-dioxbcyclohex-lylidene)ethylaminoj-3,4,6-tri-0- acetyl-P-D-glucopyranoside (4) 2-Deoxy-2-il-(4,4-dimethyi-2,6-dioxocyclohex-lylidene)ethylamino]-3,4,6-tri-O-acetyl-X-D- glucopyranosyl bromide (60 mg, 0.11 mmcl) was dissolved in CH 2 Cl2 (5 ml), cooled to -15 0 C and silver trifluoro-methanesulphonate (43 mg, 0.16 mmcl) in MeOH (1 ml) added. The reaction mixture was stirred overnight, filtered and the filtrate evaporated. The residue was washed with saturated NaHC03 solution, dried over MgSO 4 and evaporated. The residue was purified by chromatography, to give Methyl 2-Deoxy-2-[l- (4.4-dimethyl-2, 6 -dioxocyclohex-l-ylidene)ethylamino]- 3, 4 6 -tri-O-acetyl-g-D-glucopyranoside (40 mg, Rf 0.35 (Hexane/EtOAc 1:1); FAB MS C 2 3

H

3 3 N0 1 0 (483.49) m/z 506 484 (100), 442 1 H NMR (CDCl 3 8 13.84 1H, NH), 5.20 1H, H-3), 5.09 1H, 4.41 1H, H-1, J 1 2 =8.29 Hz), 4.32 (dd, 1H, 4.14, 3.94 (2m, 2H, 3.75 1H, 3.48 3H, OCH 3 2.57 3H, CH 3 2.37 4H, 2 CH2), 2.09, 2.03, 1.96 (3s, 9H, 3 AcO), 1.02 6H, 2 CH 3 ),and Me thyl 2-Deoxy-2- 4-dimethyl-2, 6-dioxocyclohex-1yl idene)ethylaaminoj- 3,4, 6 -tri-O-acetyl-a -D-glucopyranoside (4 (3 mg, 6-Qo Rf 0.33 (Hexane/EtOAc 1:1); FAB MS C 2 3

H

3 3 N0 1 0 (483.49) m/z 506 484 [M+HI+ (100).

WO 98/39197 PCT/AU98/00131 15 'H NMR (CDCl 3 8 13.55 1H, NH) 5.40 1H, H-3), 5.08 Ct, 1H, 4.82 1H, H-l,.J 1 2 =3.37 Hz), 4.32 (dd, 1H, 4.12 Cm, 3H, H-6, 3.53 3H,

OCH

3 2.58-(s, 3H, CH 3 2.41 Cs, 4H, 2 CH 2 2.11, 2.02, 1.94 (3s, 9H. 3 AcO), 1.02 Cs, 6H, 2 CH 3 Example 5 Synthesis of Dde-protected aminosugar uroniun salts S-[2-Deox-y-2-f1- 4-dimethyl-2, 6-dioxocyclohex-l-ylidene) ethyl aminoj 6 -tri-O-acetyl-p-D-glucopyranosylJ isothiouroniurn bromide Thiourea (14 mg, 0.18 mmol) was added to a solution of 2 -Deoxy-2--[1-C4,4-dimethy1-2,6-dioxocyclohex-lylidene) ethylamino] 6-tri-O-acetyl-cz-D-glucopyranosyl bromide (100 mg, 0.18 minol) in acetone C0.5 ml). The mixture was ref luxed for 15 min then evaporated. The residue was purified by chromatography using CHC1 3 /MeOH 5:1 as the mobile phase to give S-[2-Deoxy-2-[1-(4,4-dimethyl- 2, 6-dioxocyclohex-l-ylidene)ethylamino) 6-tri-O-acetylf9-D-gluco-pyranosyllisothiouroniun bromide Rf 0.46 CCHCl 3 /MeOH 5:1); FAB MS C 23

H

34

N

3 0 9 S (608.42) m,/z M% 528 [M-Brlj+ 452 (100).

1H NMR (CDCl 3 8 13.85 1H, NH), 5.30 1H, H-3), 5.12 1H, 4.75 1H, H J 1 2 =9.43 Hz), 2.62 3H, CH 3 2.36 4H, 2 CH 2 2.11, 2.04, 1.96 (3s, 9H, 3 AcO), 1.02 Cs, 6H, 2 CH 3 WO 98/38197 PCT/AU98/00131 -16 Example 6 Synthesis of Dde-protected alkyithiolated amino sugars Methyvl 2-Deoxy-2-[-(4, 4-dimethyl-2, 6-dioxocyclohex-lylidene) ethylaminoj-l-thio-3, 4,6- tri-O-acetyl-P3-Dglucopyranoside (6) 2-Deoxy-2-[l-(4,4-dimethyl-2, 6-dioxocyclohex-lglucopyranose (72 mg, 0.148 minol) was dissolved in acetone (0.15 ml) and K 2 C0 3 (23 mg) in water (0.15 ml) added. The reaction mixture was stirred under N 2 at room temperature and methyliodide (23 mg, 0.163 mmol) added. After 30 min stirring the reaction mixture was concentrated under reduced pressure. CH 2 Cl 2 (2ml) was added to the reaction mixture and the layers were separated. The organic phase was washed with water (0.5 ml), dried over MgSO 4 and evaporated. The residue was purified-by chromatography using EtOAc/hexane 3:1 to give Methyl 2-Deoxy-2-[l-(4,4dimethyl-2, 6-dioxocyclohex-1-ylidene)ethylamino] -1-thic- 3,4,6-tni- O-acetyl-p3-D-glucopyranoside (50 mg, 67%).

Rf 0.41 (EtOAc/hexane 3:1); FAB MS C 23

H

33 N0 9 S (499.49) m/z M% 522 500 (100), 452 338 1 H NMR (CDC1 3 8 13.96 1H, NH), 5.2'2 1H, H-3), 5.13 1H, 4.61 1H, H-1, J 1 2 =9.98 Hz), 4.30 (dd, lH, 4.15 (in, 2H, 2.60 3H, CH 3 2.42 4H, 2 CH 2 2.20 3H, SCH 3 2.09, 2.02, 1.96 (3s, 9H, 3 AcO), 1.03 6H, 2 CH 3 Example 7 Synthesis of Dde-protected benzylated aminosugars Benzyl 2-Deoxy-2-fl- 4-dimethyl-2, 6-dioxocyclohex-lylidene) ethylaminoj glucopyranoside (7) A solution of Benzyl 2-Acetamido-2-deoxy-a-Dglucopyranoside (4.70 g, 15.11 rnmol) in 1 M NaOH solution WO 98/38197 PCT/AU98/00131 17 was ref luxed at 120 0 C for 15 h. The reaction mixture was cooled to room temperature, neutralised with 1 M HCl solution and concentrated. The residue was dissolved in dry EtOH (50 ml) and filtered. 2-Acetyldimedone (4.11 g, 22.6 inmol) ah-d N,N- diisopropylethylamine (2 ml) were added to the filtrate, a nd the mixture was ref luxed for 2 h. The reaction mixture was evaporated to dryness, and the residue was taken up in EtOAc (50 ml), washed with 1M KHS0 4 solution, brine, and evaporated to give Benzyl 2-Deoxy-2- 4 4 -dimethyl-2,6-dioxocyclohex-1-ylidene)ethylamino] -a- D-glucopyranoside (3.78 g, 58%).

Rf 0.43 (CH 2 Cl 2 /EtOAc/MeOH 10:7:3); FAB MS C 23

H

31 N0 7 (433.48) m/z M% 456 434 (100), 452 338 1HNMR (CDCl 3 8 13.44 1H1, NH), 7.33 7.21 (in, 511, Ar-H), 4.80 1H, H-1, J 1 2 =3.45 Hz), 4.71, 4.56 (2d, 211, CH 2 Ar), 2.45 3H, CH 3 2.31 4H, 2 CH 2 0.99 6H, 2 CR 3 Example 8 Synthesis of Dde-protected azido derivative of arninosugars 2-Deoxy -2-fl- 4-dimethyl-2, 6-dioxocyclohex-l-ylidene) ethylainino) 6 -tri-O-acetyl-(o-D-glucopyranosy1 azide (8) A mixture of 2-Deoxy-2-[1-(4,4-dimethyl-2,6dioxocyclohex-l-ylidene) ethylaminol glucopyranosyl bromide (100 mg, 0.18 mmnol), sodium azide 100 mg, 1.56 inmol) in DMF (5 ml) was stirred at 80 0 C for 2 hours. The reaction mixture was evaporated, taken up in

CH

2 Cl 2 (10 ml), washed with 1120 (2 x 2 ml), dried over MgSO 4 and concentrated. The residue was purified by chromatography, using hexane/EtOAc 1:1 as the mobile phase, to give 2-Deoxy-2-[l- (4,4-dirnethyl-2, 6-dioxocyclohex-lylidene) ethylamino] 6 -tri-o-acetyl-o-D-glucopyranosyl azide (65 mig, WO 98/38197 PCT/AU98/00131 -18- Rf 0.55 (hexane/EtOAc 1:1); FAB MS C 22

H

30

N

4 0 9 (494.48) m/z M% 517 495 [M+HV+ (100); 452 338 1NMR (CDCl 3 13.91 1H, NHl), 5.19 Ct, 1H, H-3), 5.10 1H, 4.87 1H, H-1, J 1 2 =8.95 Hz), 4.34 (dd, 1H, 4.15 (dd, 1H, H-61), 3.85 (in, 2H, 2.59 Cs, 3H, CH 3 2.38 Cs, 4H, 2 CH 2 1.02 Cs, 6H, 2 CH 3 Example 9 Synthesis of Dde-protected thiolated aminosugars 2-Deoxy-2-f 1- 4-Dimethyl 6-dioxocyclohex-1-ylidene) ethyl amino] -l -thio-3, 4, 6-tri-Q- acetyl-P~-D-glucopyranose (9) To S-12-Deoxy-2-[1-C4,4-dimethyl-2,6dioxocyclohex-1-ylidene) ethylamino] 6-tri-O-acety1-p-Dglucopyranosyl]isothiouronium bromide (136 mng, 0.22 inmol) a solution of Na 2

S

2

O

5 (43 mng, 0.225 iniol) in water (0.2 ml) and 1,2-dichioroethane (0.24 ml) was added. The reaction mixture was kept under ref lux at 85 0 C for 20 min. After dilution with CH 2 C1 2 (5 ml), the layers were separated, the organic phase was washed with water (3 ml), dried over MgSO 4 concentrated under reduced pressure, and chroinatographed using ether /MeOH 10:1 to give 2-Deoxy-2- 4-dimethyl-2, 6-dioxocyclohex-l-ylidene) ethylamino] -1thio-3,4, 6 -tri-O-acetyl-p3-D-glucopyranose (95 mg, 87%).

Rf 0.31 Cether/MeOH 10:1); FAB MS C 22

H

31 N0 9 S (485.47) m/z M% 508 [M-iNa]+ 486 [M+Hh1+ (100), 452 338 IHNMR CCDCl 3 8 13.97 Cd, 1Hl, NH), 5.32 Ct, IH, H-3), 5.15 Ct, l1H, 4.75 Cdd, 1Hl, H-1, J1,2=8.29 Hz), -WO 98/38197 PCT/AU98/00131 -19 3.85 (in, 1H, H- 2.62 3H, GCl 3 2.38 4H, 2 CH 2 2.10, 2.04, 1.96 (3s, 9H, 3 AcO), 1.02 6H, 2 CH 3 Example 10 Synthesis of Dde-protected benzylidene derivative of arninosugars Benzyl 4, 6-Q-Benzylidene-2-deoxyv-2- 4-dimethyl-2, 6di oxocycl ohex- yl iden e) ethyvl aminocI -aL -D -gl u copyranosi de A mixture of benzaldehyde (1 ml), formic acid (1 ml) and Benzyl 2-Deoxy-2-[l-(4,4-dimethyl-2,6dioxocyclohex-1-ylidene) ethylarnino] -a-D-glucopyranoside (433 mng, 1 minol) was stirred at room temperature for 2 h.

The reaction mixture was evaporated to dryness using a high vacuum rotary evaporator. The residue was treated with ether (40 ml) and the suspention filtered. The solid purified by chromatography, using CHCl 3 -EtOAc 10:4 as the mobile phase, to give Benzyl 4,6-O-Benzylidene-2-deoxy-2- 4-dimethyl-2, 6-dioxocyclohex-l-ylidene) -ethylamino] c-D-glucopyranoside (10) (340 mg, RfO.38 (CHCl 3 -EtOAc 10:4); FAB MS C 30

H

35 N0 7 (521.58) m/z M% 544 522 (100), 338 1NMR (CDC1 3 8 13.52 1H, NH), 7.37 7.26 (in, Ar-H), 5.56 1H, GCl-Ar), 4,90, 4.60 (2d, 2H, CH 2 -Ar), 4.79 1H, H-1, J 1 2 =3.08 Hz), 4.35 1H, H-4), 4.26 (dd, 1H, 3.98 (mn, 2H, H-5, 3.77 1Hl, Hl-61), 3.63 1Hl, 2.57 3H, CH 3 2.33 4H, 2 CH 2 1.01 6H, 2 CH 3 WO 98/38197 PCT/AU98/00131 Example 11 Synthesis of Dde protected reducing arninosugars 2-Deoxy-2-[l- 4-dimethyl 6-dioxocyclohex-1-_ylidene) eth!ylamino) 6-tn -0-acetyl -cz-D-gl ucopyranose (12) Benzyl 2-Deoxy-2-[l-(4,4-dimethyl-2,6dioxocyclohex-l-ylidene) ethiylamino] -a-D-glucopyranoside (400 mg, 0.92 mrnol) was dissolved in. pyridine (6 ml) and cooled to 0 0 C, then acetic anhydride (10 ml) was added dropwise. The solution was stirred at room temperature overnight, then evaporated. The residue was purified by chromatography using EtOAc/hexane 3:1 to give Benzyl 2- Deoxy-2- 4-dimethyl-2, 6-dioxocyclohex-l-ylidene) ethylamino] 6-tri-o-acetyl-a-D-glucopyranoside (11) (465 mg, Rf 0.41 (EtOAc/hexane 3:1); FAB MS C 2 9

H

37 N0 10 (559.59) rn/z M% 532 560 (100), 452 338 1 H NMR (CDCl 3 8 13.66 1H, NH), 7.43 7.32 (mn, Ar-H) 5.45 1H, 5.07 1H, 4.93 1H, H-1, J 1 2 =3.53 Hz), 4.76, 4.72 (2d, 2H, CH 2 4.29 (dd, lH, 4.07 (mn, 2H, H-61, H-5),'3.96 (dd, 1H, H-6), 2.52 3H, CH 3 2.38 4H, 2 CH 2 2.10, 2.00, 1.94 (3s, 9H, 3 AcO), 1.03 6H, 2 CH 3 Benzyl 2 -Deoxy-2-[1-(4, 4-dirnethyl-2,6-dioxocyclohex-l-ylidene) ethylainino] 4, 6-tri-O-acetyl-a-Dglucopyranoside (11) (100 mg, 0.17 mmol) was dissolved in MeOH (5 ml) and'hydrogenated over Pd/C (20 mg) overnight. The suspension was filtered, and the filtrate was evaporated to give 2-Deoxy-2--[l-(4,4-dimethyl-2,6dioxocyclohex-1-ylidene) ethylamino] -3 6-tri-o-acetyl-a-Dglucopyranose (12) (75 mg, Rf 0.44 (CHCl 3 /EtOAC 1:1); WO 98/38197 PCV/AU98/00131 21 FAB MS C 22

H

3 1 N0 10 (469.47) r/z 492 470 (100), 452 1 H NMR (CDC1 3 5 13.81 1H, NH), 5.49 1H, H-3), 5.28 1H, H-1, J 1 2 =3.29 Hz), 5.11 1H, H-4), 4.42 Cdd, H, 4.33 (dd, H, 2.59 3H, CH 3 2.37 4H, 2 CH 2 2.10, 2.03, 1.96 (3s, 9H, 3 AcO), 1.01 6H, 2 C- 3 Example 12 Synthesis of Dde-protected trichioroacetimidate of aminosugars 2 -Deoxy-2 [1 4 -dinethyl 6-di oxocycl ohex-1 -yl idene) ethylaamino-3,4, 6 -tri-O-acetylcx,-D-glucopyranosyl trichloroacetimidate (13) A mixture of 2-Deoxy-2-[1-(4,4-dimethyl-2,6dioxocyclohex-1-ylidene)ethylamino]-3,4,6-tri-O-acetyl-a-Dglucopyranose (100 mg, 0.21 mmol) and trichloroacetonitrile in CH 2 C1 2 was cooled to OOC and 1,8-diazabicyclo(5.4.0)undec-7-en (2 mg) added. The reaction mixture was stirred at OOC for 1.5 h and at room temperature for 2 h. The solution was evaporated, and the residue chromatographed using CHCl 3 /EtOAc 1:1 as the mobile phase to give 2-deoxy- 2-[1-(4,4-dinethyl-2,6-dioxocyclohex-l-ylidene)ethylamino]- 3,4, 6-tri-O-acetyl-a, P-D-glucopyranosy1 trichloroacetimidate (13) (71 mg, Rf 0.61 (CHCl 3 /EtOAc 1:1); FAB MS C 24

H

31 C1 3

N

2 0 1 0 (613.88) m/z 635 452 (100).

1H NMR (CDC1 3 8 13.95, 13.72 (2d, 1H, NHA), 8.84, 8.76 (2s, 1H, NHA), 6.48 H-la, J 1 2 3.05 Hz), 5.85 H-ljS, J 1 2 =8.72 Hz), 5.52 1H, 5.31 (t, 1H, 2.65, 2.63 (2s, 3H, CH3aj,S), 2.31 (2s, 4H, WO 98138197 PCT/AU98/00131 22 2 CH2a,), 2.09, 2.08, 2.05, 2.04, 1.99, 1.97 (6s, 9H1, 3 AcOa,S), 0.99, 0.98 (2s, 6H, 2 CH3(X,).

Example 13 Synthesis of Dde-protected 0-triphenylmethylated aminosugars Ben zyl 2 -Deoxy-2 4-dirnethyi 6-di oxocyclohex-1 yVlidene) ethyilaminoj -6-0-triphenylmethyl -c-D-giucopyranoside (14) A mixture of Benzyl 2-Deoxy-2-[1-(4,4-dimethyl- 2, 6-dioxocyclohex-1-ylidene) ethylamino] -a-D-glucopyranoside (100 mg, 0.23 mmol), triphenylmethyibromide (149 mg, 0.46 mmol) in DMF/pyridine 1:1 (2 ml) was stirred at 100 0

C

for 15 h. The reaction mixture was evaporated, the residue was taken up in CHC1 3 (10 ml), washed with water (3 ml), dried over MgSO 4 and concentrated. The residue was purified by chromatography using CHC1 3 /MeOH 10:1 as the mobile phase to give Benzyl 2-Deoxy-2-[1-(4,4-dimethyl-2, 6dioxocyclohex-l-ylidene) ethylamino] -6-O-triphenylmethyl-(X- D-glucopyranoside (14) (104 mg, 64%).

Rf 0.55 (CHC1 3 /MeOH 10:1); FAB MS C 42

H

45 N0 7 (675.68) m/z M% 698 676 (100) 1NMR (CDC1 3 8 13.49 1H, NH), 7.49 7.23 (in, Ar-H), 4.87, 4.66( 2d, 2H, CH 2 Ar), 4.83 1H, H-i,

J

1 2 =3.70 Hz), 3.84 1H, 2.55 3H, CH 3 2.31 4H, 2 CH 2 1.02 6H, 2 CE! 3 Example 14 Synthesis of Dde-protected 0-silylated aminosugars Ben zyl 2 -Deoxy-2 4-dimethyl 6-di oxocycilohex-1 ylidene) ethyl aminoJ -6-O-t -butyldimethyvlsilyvl -c-Dglucopyranoside Benzyl 2-Deoxy-2-[1-(4,4-diinethyl-2,6dioxocyclohex-l-ylidene) ethylamino] -a-D-glucopyranoside WO 98/38197 PCT/AU98/00131 23 (100 mg, 0.23 mmol) was dissolved in dry pyridine (2 ml), cooled to 0°C and t-butyldimethylsilylchloride (39 mg, 0.26 mmol) added. The reaction mixture was stirred at room temperature overnight. The solution was evaporated, the residue was taken up in CHC1 3 (10 ml), washed with water (3 ml), dried over MgSO 4 and concentrated. The residue was purified by chromatography using CHC1 3 /MeOH 10:1 as the mobile phase to give Benzyl 2-Deoxy-2-[1-(4,4-dimethyl-2,6dioxocyclohex-1-ylidene)ethylamino]-6-O-t-butyldimethylsilyl-a-D-glucopyranoside (15) (77 mg, 61%).

Rf 0.57 (CHC1 3 /MeOH 10:1); FAB MS C 2 9

H

4 5 N0 7 Si (547.74) m/z 570 548 (100).

1H NMR (CDC1 3 6 13.45 1H, NH), 7.40-7.27 Ar-H), 4.88, 4.65( 2d, 2H, CH 2 Ar), 4.79 1H, H-l,

J

1 2 =3.42 Hz), 2.55 3H, CH 3 2.31 4H, 2 CH 2 1.02 6H, 2 CH 3 0.93 9H, 3 CH 3 0.10 6H, 2 CH 3 Si).

Example 15 Synthesis of partially protected polyaminosugars 2-Deoxy-2-[l- 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethylamino]-3,4, 6 -tri-O-acetyl--D-glucopyranosyl amine (16) 2-Deoxy-2-[1-(4,4-dimethyl-2,6-dioxocyclohex-1ylidene)ethylaminol-3,4, 6 -tri-O-acetyl--D-glucopyranosyl amine (60 mg, 0.12 mmol) was dissolved in MeOH (5 ml) and hydrogenated over Pd/C (10 mg) overnight. The suspension was filtered, the filtrate was evaporated to give 2-Deoxy-2-[1-(4,4-dimethyl-2,6-dioxocyclohex-lylidene)ethylamino]-3,4, 6 -tri-O-acetyl-P-D-glucopyranosyl amine (16) (45 mg, Rf 0.38 (EtOAc); WO 98/a8I97 PCT/AU98/00131 -24 FAB MS C 2 2

H

3 2

N

2 0 9 (468.50) m/z M% 491 (100), 469 452 1 H NMR (CDC13) 8 13.75 1H, NH), 2.61 3H, CH 3 2.35 4H, 2 CH 2 2.09, '2.02, 1.98 (3s, 9H, 3 AcO), 1.03 6H, 2 CH 3 Example 16 Synthesis of Dmab-protected sugars 4 -dime thyl 6-cioxocyclohexylidene) -ethyl]jamino]benzyl (1,2,3,4-tetra-O-acetyl-f3-D-glucop-yranose) uronate (17) A mixture of 1,2,3,4-tetra-O-acety1-P-Dglucuronic acid (100 mg, 0.27 inmol), 4-[N-[l-(4,4-dimethyl- 2, 6-dioxocyclohexylidene)ethyllaminolbenzyl alcohol (79 mg, 0.27 mmol), 1,3- dicyclohexylcarbodiimide (62 mg, 0.30 inmol) in CH 2 C1 2 was stirred overnight at room temperature. The reaction mixture was evaporated, the residue was purified by chromatography using CHC1 3 /EtOAc 10:4 to give 4-[N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene) ethyl] -aminolbenzyl 4-tetra-O-acetyl-3-Dglucopyranose)uronate (17) (92 mg, 53%).

Rf 0.51 (CHC1 3 /EtOAc 10:4); FAB MS C 31

H

37 N0 13 (631.61) m/z M% 654 632 270 (100).

INMR (CDC1 3 5 15.06 1H, NH) 7.41 2H, 2 Ar-H) 7.15 2H, 2 Ar-H), 5.76 1H, H-1, J 1 2 =9.08 Hz), 4.22 1H, H-5, J 1 2 =9.36 Hz), 2.51 3H, CH 3 2.37 4H, 2 CH 2 2.09, 2.00, 1.86 (3s, 9H, 3 AcO), 1.07 6H, 2 CH 3 WO 98/38197 PCT/AU98/00131 Example 17 Synthesis of Dde- and N-acyl-protected polyaminosugars 2-Acetamido-3,4, 6-tri -O-acetyl-1,2-dideoxy-1-f1-(4, 4dimethyl-2, 6-dioxocyclohex-1-ylidene) ethyl amino] -P -Dglucopyranose (19) 2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-p-Dglucopyranosyl azide (100 mg, 0.26 iniol) was dissolved in MeOH (5 ml) and hydrogenated over Pd/C (10 ig) for h. The suspension was filtered, and the filtrate was evaporated to give 2-Acetaiido-2-deoxy-3,4,6-tri-O-acetyl- P-D-glucopyranosyl amine (18) (80 mg, 86%).

Rf 0.38 (CHCl 3 /MeOH 10:1); FAB MS C 14

H

22

N

2 0 8 (346.34) m/z 347 (100), 330 IH NMR (CDCl 3 8 5.64 1H, NH), 3.99 1H, H-2), 3.65 1H, 2.11, 2.04, 2.02, 1.97 (4s, 12H, 3 AcO, AcNH).

A mixture of 2-Acetaiido-2-deoxy-3,4,6-tri-0acetyl-P-D-glucopyranosyl amine (80 mg, 0.23 mmol) and 2-acetyldiiedone (55 mg, 0.30 mmol) in MeOH (5 ml) was refluxed for 5 h. The reaction mixture was evaporated, the residue was purified by chromatography using CHCl 3 /MeOH 10:0.5 as the mobile phase, to give 2-Acetanido-3,4,6-tri- O-acetyl-1,2-dideoxy-l-[l-(4,4-dimethyl-2,6-dioxocyclohexl-ylidene)ethylaino] -j-D-glucopyranose (19) (70 ig, Rf 0.37 (CHCl 3 /MeOH 10:0.5); FAB MS C 24

H

34

N

2 0 10 (510.53) m/z 533 [M+Nal+ 511 (100), 330 1H NMR (CDCl 3 5 13.60 1H, NH), 5.81 1H, NH), 5.45 1H, 5.31 1H, 5.05 1H, H-4), WO 98/38197 PCT/AU98/00131 -26 4.21 (dd, 1H, 4.11 (dd, 1H, 3.92 (mn, 1H, H-2), 3. 82 (mn, 1H, H-5) 2.58 3H, CH 3 2 .35 4H, 2 C2 2.06, 2.04, 2.02, 1.92 (3s, 9H, 2 AcO, AcNH), 1.01 6H, 2 CH 3 Example 18 Synthesis of Dde-protected 0-isopropylidene derivative of aminosugars Benzyl1 2-Deoxy-2- 1- 4-dimethyl-2, 6-dioxocyclohex-l- _ylidene) ethyl arinoj 6-O-isopropyl iden e-i-Dglucopyranoside A mixture of Benzyl 2-Deoxy-2-[1--(4,4-dirnethyl- 2, 6-dioxocyclohex-1-ylidene) ethylamino] -aX-D-glucopyranoside (100 mng, 0.23 mniol) and (+/-)-l0-camphorsulphonic acid mng) in 2,2- dimethoxypropane (10 ml) was ref luxed for 2 h.

The reaction mixture was evaporated, and the residue was taken up in CH 2 C1 2 (10 ml), washed with saturated NaHCO 3 solution (3 ml), and concentrated. The residue was purified by chromatography using CH 2 C1 2 /MeOH 10:1 as the mobile phase to give Benzyl 2-Deoxy-2-[1-(4,4-dimethyl-2,6dioxocyclohex-1-ylidene) ethylamino] 6-O-isopropylidene-z- D-glucopyranoside (20) (82 mg, Rf 0.44 (CH 2 C1 2 /MeOH 10:1); FAB MS C 26

H

35 N0 7 (473.54) m/z M% 496 [M+NaI+ 474 (100), 382 1H NMR (CDC1 3 8 13.48 1H, NH), 7.38 7.27 (mn, Ar-H), 4.97, 4.65( 2d, 2H, CH 2 Ar), 4.76 1H, H-i,

J

1 2 =3.55 Hz), 2.55 3H, CH 3 2.31 4H, 2 CH 2 1.52, 1.30 (2s, 6H, 2 CH 3 1.00 6H, 2 CH 3 WO 98/38197 PCT/AU98/00131 27 Example 19 Synthesis of Dde- protected galactoaminosugars 2-Deoxy-2-- [(4,4-dimethyl-2, 6-dioxocyclohex-1-ylidene)ethylamino]-D-galactopyranose (21) Sodium (22 mg, 0.95 mmol) was added to abs.

methanol (10 ml) and the reaction mixture was stirred for min. D-galactosamine hydrochloride (206 mg, 0.95 mmol) was added to the resulting clear solution, and the reaction mixture was stirred at room temperature for another 5 min.

2-Acetyldimedone (261 mg, 1.43 mmol) was added and the reaction mixture was stirred under reflux for 5 hours. The solution was cooled and the product was precipitated by ether (100 ml) resulting in 2-Deoxy-2-[1-(4,4-dimethyl-2,6dioxocyclohex-1-ylidene)ethylamino]-D-galactopyranose (21) (270 mg, Rf 0.37 (MeCN/H 2 0 10:0.5); FAB MS C 16

H

25 N0 7 (343.33) m/z 366 [M+Na] 344 (100), 327 1H NMR (D 2 0) 6 5.34 H-lA, J 1 3.54 Hz), 4.87 H- 1g), 4.28 (dd, H-2a), 4.17 H-2g), 4.08 H-4a), 4.03 H-4g), 2.56 3H, CH 3 2.48, 2.44 (2s, 4H, 2

CH

2 1.03 6H, 2 CH 3 Example 20 Synthesis of Nde-protected aminosugars 2-Deoxy-2-[l-(4-nitro-1,3-dioxoindan-2-ylidene)-ethylamino]-D-glucopyranose (22) Sodium (126 mg, 5.47 mmol) was added to abs.

methanol (50 ml) and the reaction mixture was stirred for min. D-glucosamine hydrochloride (1.18 g, 5.47 mmol) was added to the resulting clear solution and the reaction mixture was stirred at room temperature for another 5 min.

2- acetyl-4-nitroindane-1,3-dion (1.91 g, 8.21 mmol) was added and the reaction mixture was stirred under reflux for hours. The solution was cooled and the product was WO 98/38197 PCT/AU98/00131 -28 filtered of f. The solid was washed with MeOH (10 ml), ether (50 ml) and dried, affording 2-Deoxy-2-[1-(4-nitro- 1,3- dioxoindan-2-ylidene) ethylamino] -D-glucopyranose (22) (1.10 g, Rf 0.41 (MeCN/H 2 0 10:0.5); FAB MS C 17

H

18

N

2 0 9 (394.32) m/z M% 395 [M+HP+ (100).

1 H MR (D 2 0) 8 7.75-7.40 (in, 3H, 3 Ar-H), 5.21 H-la), 3.95-3.25 (sugar 6H), 3.18 3H, CH 3 Example 21 Synthesis of Nde protected 0-acetylated aminosugars 2 -Deoy- 2 -fl-( 4 -nitro-1,3-dioxoindan-2>ylidene) ethyl amino] 6 -tri-O-acetyvl-aX-D- glucopyranose (23) A mixture of 2-Deoxy-2-[l-(4-nitro-1,3dioxoindan-2-ylidene) ethylamino] -D-glucopyranose (100 mg, 0.23 inmol), pyridine (2 ml) and acetic anhydride (3 ml) stirred at room temperature overnight. The reaction mixture was evaporated, and the residue was purified by chromatography using CHCl 3 /EtOAc 10:4 as the mobile phase to give 2-Deoxy-2- rl-(4-nitro-l,3-dioxoindan-2-ylidene) ethylamino] 3 4 6 -t ri-O-acetyl-aX-D-glucopyranose (23) (165 mng, 79%).

FAB MS C 25

H

26

N

2 0 13 (562.48) m/z M% 585 563 (100), 503 1 H NMR (CDC1 3 5 11.00, 10.90 (2d, 1H, NHEZ), 7.95-7.68 (mn, 3H, 3 Ar-H), 6.25, 6.24 (2d, 1H, H-lE,Z), 5.43 1H, 5.18 1H, 2.68 3H, CH 3 2.38, 2.07, 2.04, 2.00 (4s, 12H, 4 AcO).

WO 98/38197 PCT/AU98/00131 29 Example 22 Synthesis of Dde-protected deoxyaminosugars with furanose ring 3 '-deoxy-3'-[1 4-dimethyl-2, 6-dioxocyclohex-1-ylidene)ethylamino]-thymidine (24) 3 '-Deoxy-3'-azido-thymidine (200 mg, 0.75 mmol) was dissolved in MeOH (25 ml) and Pd/C (40 mg) was added.

The suspension was stirred over a constant stream of H 2 overnight. The reaction mixture was filtered, and the filtrate was concentrated. The residue was taken up in abs. EtOH (5 ml), N,N-diisopropylethylamine (0.1 ml) and 2-acetyldimedone (204 mg, 1.12 mmol) were added and the solution was refluxed for 5 h. The reaction mixture was cooled to room temperature and the product was precipitated by adding ether (50 ml) giving 3'-deoxy-3'-[1-(4,4dimethyl-2, 6 -dioxocyclohex-1-ylidene)-ethylamino]-thymidine (24) (200 mg, 66%).

Rf 0.45 (CH 2 C1 2 /EtOAc/MeOH 10:7:3); FAB MS C 20

H

27

N

3 0 4 (405.45) m/z 428 406 (100).

1H NMR (CDC1 3 8 13.79 1H, NH), 7.55 1H, H-6), 6.13 1H, 4.70 1H, 4.04 1H, 3.96 (dd, 1H, H- 5 3.72 (dd, 1H, H- 5 2.55 3H,

CH

3 2.42 1H, 2.32 4H, 2 CH 2 1.80 (s, 3H, CH 3 0.96 6H, 2 CH 3 Example 23 Synthesis of Dde-protected aminosugar containing oligosaccharides 6 -tetra-O-acetyl-a-D-galactopyranosyl)-2,3, 6tri-O-acetyl-N-[1-(4,4-dimethyl-2,6-dioxocyclohex-lylidene)ethyl ]--D-glucopyranosyl amine (27) A mixture of -lactose octaacetate (203 mg, 0.3 mmol), trimethylsilyl azide (41 mg, 0.35 mmol), and SnCl 4 (40 mg,0.15 mmol) in CH 2 C1 2 (1.5 ml) was stirred overnight at room temperature. The solution was diluted WO 98/38197 PCT/AU98/00131 with CH 2 C1 2 (20 ml) and washed twice with 1 M potassium fluoride solution (5 ml), water (5 ml) and evaporated affording pyranosyl) 6 -tri-0-acetyl-j3-D-glucopyranosyl azide (178 mg Rf 0.38 (hexane/EtOAc 1:1); FAB MS C 26

H

35

N

3 0 17 (661.56) m/z M% 684 [M+Na]l 662 331 (100).

1NMR (CDCl 3 5 5.35 1H, 4.95 1H, H-11,

J

1 2 =3.63 Hz), 4.61 1H, H-1, J 1 2 =9.13 Hz), 2.14, 2.13, 2.07, 2.06, 2.04, 1.96 (6s, 21H, 7 AcO).

2 3 4 6 -tetra-0-acetyl-aX-D-galactopyranosyl) 6 -tri-0-acetyl-p-D-glucopyranosyl azide (178 mg, 0.26 inmol) was dissolved in MeOH (5 ml) and hydrogenated over Pd/C (10 mg) for 5 h. The suspension was filtered, and the filtrate was evaporated to give 4-0- 6 -tetra-O-acetyl-cz-D-galacto-pyranosyl) 2,3, 6 -tri-0-acetyl-3-D-glucopyranosyl amine (26) (157 mg, 92%).

Rf 0.41 (EtOAc); FAB MS C 26

H

37 N0 17 (635.56) m/z M% 658 [M-iNa]+ 636 331 (100).

1 H NNP. (CDCl 3 8 5.35 1H, 2.15, 2.12, 2.07, 2.06, 2.04, 2.03, 1.96 (7s, 21H1, 7 AcO).

A mixture of 3,4, galactopyranosyl) 3, 6 -tri-O-acetyl-J3-D-glucopyranosyl amine (157 mg, 0.24 mmol) and 2-acetyldimedone (81 mg, 0.45 inmol) in MeOH (5 ml) was ref luxed for 5 h. The reaction mixture was evaporated, and the residue was purified by W.0 98/38197 PCT/AU98/00131 -31 chromatography using CHC1 3 /EtOAc 1:1 as the mobile phase, to give 4-0- 6-tetra-O- acetyl-ax-D-galactopyranosyl) 2,3, 6-tri-O-acetyl-N-[1- 4-dimethyl-2, 6-dioxocyclohex-1ylidene) ethyl] -0-D-glucopyranosyl amine (27) (106 mg, 54%).

Rf 0.39 (CHCl 3 /EtOAc 1:1); FAB MS C 36

H

49 N0 19 (799.75) m/z M% 822 (50) 800 (100).

IH NMR (CDC1 3 5 13.56 1H, NH) 5.35 1H, H-i'

J

1 2 =3.13 Hz), 2.60 3H, CH 3 2.36 4H, 2 CH 2 2.15, 2.12, 2.07, 2.06, 2.04, 2.03, 1.96 (7s, 21H, 7 AcO), 1.02 6H, 2 CH 3 Example 24 Synthesis of 2 -Acetyl-4-nitroindan-1,3-dione 2-Ace tyvl-4 -nitroindan -1 ,3-dione A mixture of 3-nitrophthalic anydride (12 g, mmol), anhydrous pyridine (25 ml), piperidine (0.2 ml) and 2,4-pentanedione (6.25 g, 60 mmol) was stirred at 40 0

C

for 6 h. The reaction mixture was cooled to 0 0 C and the crystalline mass was collected at the pump, washed with ether, and dried to give the yellow pyridinium salt. The salt was treated with 6 M HCl (100 ml) and the solid was filtered off. The product was crystallised from isopropanol to afford 2-Acetyl-4- nitroindan-1,3-dione (8.74g, 79%).

Rf 0.44 (EtOAc/AcOH 100:0.2); FAB MS CljH 7 N0 5 (233.17) m/z M% 256 234 IIM-iH]+ (100).

1NMR (CDC1 3 5 8.09 -7.83 (in, 3H, 3 Ar- 2.62, 2.60 (2s, 3H, CH3(E,Z)).

WO 98/38197 PCT/AU98/00131 32 It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this invention.

References cited herein are listed on the following pages, and are incorporated by this reference.

WO 98/38197 PCT/AU98/00131 -33-

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Claims (15)

1. A compound containing a sugar carrying one or more primary amine groups protected with a 2-substituted- 1,3-dioxo compound of General Formula I or General Formula II: O O R1 R R RV R R 0 NO 2 0 I II in which R 1 and R 2 may be the same or different, and is each hydrogen or C1- 4 alkyl, R' is an amino sugar, a glycosylamine, or an oligosaccharide comprising at least one aminosugar or one 15 glycosylamine unit, in which the sugar is coupled via an amino group, and R" is alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl or substituted cycloalkyl.

2. A compound according to Claim 1, in which the protecting group is of General Formula I and R 1 and R 2 are both methyl.

3. A compound according to Claim 1, selected from the group consisting of Compounds 1 to 23 as described in Table 1, Compound 24 as described in Table 2 and compound 25 27 as described in Table 3.

4. A reagent for solution phase synthesis of sugar- containing compounds, comprising a cyclic 2-substituted- 1,3-dioxo compound of General Formula I or General Formula II WO 98/38197 PCT/AU98/00131 36 0 0 RI R R R 2 R' R R 0 NO 2 0 I II in which R 1 and R 2 may be the same or different, and is each hydrogen or C1- 4 alkyl, R' is an amino sugar, a glycosylamine, or an oligosaccharide comprising at least one aminosugar or one glycosylamine unit, in which the sugar is coupled via an amino group, and R" is alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl or substituted cycloalkyl.

A reagent according to Claim 4 in which the protecting group is of General Formula I and both R' and R 2 are methyl.

6. A linker-saccharide complex, comprising a linker group and a saccharide compound comprising a protecting group of General Formula I or General Formula II RIR R R R R' O NO 2 0 I II in which R 1 and R 2 may be the same or different, and is each hydrogen or C 1 4 alkyl, R' is an amino sugar, a glycosylamine, or an oligosaccharide comprising at least one aminosugar or one glycosylamine unit, in which the sugar is coupled via an amino group, WO98/38197 PCT/AU98/00131 37 and R" is alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl or substituted cycloalkyl.

7. A resin-linker-saccharide support for solid phase oligosaccharide synthesis, comprising a linker group, a resin, and a saccharide compound comprising a protecting group of General Formula I or General Formula II O 0 RI R R R 2 R" R 0 NO2 I II in which R 1 and R 2 may be the same or different, and is each hydrogen or C1- 4 alkyl, R' is an amino sugar, a glycosylamine, or an oligosaccharide comprising at least one aminosugar or one glycosylamine unit, in which the sugar is coupled via an amino group, and R" is alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl or substituted cycloalkyl.

8. A method of solution phase synthesis of oligosaccharides, comprising the step of sequentially linking mono- or oligosaccharide groups to a linker- saccharide complex as defined in Claim 6.

9. A method according to Claim 8 for synthesis of aminoglycoside compounds.

A method of solid-phase synthesis of oligosaccharides, comprising the step of sequentially linking mono- or oligosaccharide groups to a resin-linker- sugar support as defined in Claim 7.

11. A method according to any one of Claims 8 to for combinatorial synthesis. 38

12. A kit for solid-phase synthesis or combinatorial synthesis of oligosaccharides, comprising a linker- saccharide complex according to Claim 6 or a resin-linker- saccharide support according to Claim 7, and optionally also comprising one or more further reagents such as partially or differentially activated, fully protected saccharides, protecting agents, deprotecting agents, resins and/or solvents suitable for solid phase or combinatorial synthesis.

13. A compound according to claim 1, substantially as herein described with reference to the examples.

14. A linker-saccharide complex according to claim 6, substantially as herein described with reference to the examples.

15. A resin-linker-saccharide support according to claim 7, substantially as herein described with reference to the examples. Dated this 11th day of January 2001 20 ALCHEMIA PTY LTD :By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia