AU756536B2 - Protecting and linking groups for organic synthesis - Google Patents

Description

1 PROTECTING AND LINKING GROUPS FOR ORGANIC SYNTHESIS This invention relates to methods for synthesis of organic compounds, and in particular to compounds useful as protecting and linking groups for use in the synthesis of peptides, oligosaccharides, glycopeptides and glycolipids. The invention provides protecting and linking groups which are useful in both solid phase and solution synthesis, and are particularly applicable to combinatorial synthesis.

BACKGROUND OF THE INVENTION 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.

25 The problem of functional group incompatibility in the synthesis of complex organic structures demands the use of a functional group protection strategy. Complex synthetic intermediates and products usually contain a multiplicity of reactive groups, most of which must first 30 be blocked, and subsequently liberated at an appropriate point in the synthesis. The problem is especially acute in the design and construction of polyfunctional molecules such as oligosaccharides, peptides, glycopeptides and I glycolipids.

In oligosaccharide synthesis, a variety of protective groups are required. It is necessary to place groups regioselectively at specific locations; on primary \\mclb fies\homeS\suzannet\Keep\Speci\93303-98. I SPECIdoc 24/05/02 la alcohols, on cis-diols, on trans-diols, on 1,2-diols, on 1,3-diols, or on particular secondary alcohols. In addition, aminosugars are important constituents of oligosaccharides, and their amino-protection should be compatible with the hydroxy group protection strategy.

The properties of the protective group adjacent to the anomeric centre are also important. Whether this group is participating or non-participating plays a significant role in control of glycoside stereochemistry. Because most reactions at the glycosidic centre proceed via electron deficient intermediates, electron-releasing substituents on the C-2 substituent accelerate the reaction at the o \\mclb csomc$suzannct\KepSpccf93303-98. I SPECldoc 24/05/02 WO 99/15510 PCT/AU98/00808 -2glycosidic centre. Electron-withdrawing substituents, normally esters or amides, slow the reaction. In solid phase oligosaccharide synthesis, the stability and sensitivity of the linker between the first sugar unit and the resin becomes a crucial part of the protection plan.

The presence of other functional groups, such as alkenes or esters, or features such as a furanose ring in the target oligosaccharide, may dictate that the protecting groups used for the synthesis are not sensitive to acid, base, reductive, or other commonly used cleavage techniques. The choice of protecting groups is therefore one of the decisive factors in the successful realization of solid phase oligosaccharide synthesis.

In solid phase peptide and glycopeptide synthesis the demand of a new orthogonal protective set is significant. The established orthogonal deprotection sets are based upon the well-known Fmoc and Boc protection of amino acids. The construction of complex peptides or glycopeptides often requires a third orthogonal protecting group for side-chain amino functionalities, whose removal will not affect the protecting groups in the other orthogonal sets, or vice versa.

Many protecting groups have been developed for amino group protection, and fall into seven broad classes.

1. N-Acyl Derivatives a) Phthalimides are especially useful in the protection of amino functions in aminoglycoside synthesis (Nicolaou et al, 1992), because they are stable during the glycosylation, and because they help to control the stereochemistry by neighbouring group participation.

Unfortunately, the deprotection needs vigorous conditions, which often results in partial product decomposition.

b) Trifluoroacetamides (Weygand and Czendes, 1952) Simple amide derivatives are usually worthless as protecting groups because the conditions required to remove them are too harsh. However, the trifluoroacetamide group WO 99/15510 PCT/AU98/00808 -3is exceptionally labile to base hydrolysis, and is therefore useful in the protection of amines.

c) Carbamates are used as protective groups for amino acids to minimize racemization in peptide synthesis. Racemization occurs during the base-catalysed coupling reaction of an N-protected, carboxyl-activated amino acid, and takes place via the intermediate oxazolone that forms readily from an N-acyl protected amino acid.

Many carbamates, for example Boc (McKay and Albertson, 1957), Cbz (Bergman and Zervas, 1932), Alloc (Kunz and Unverzagt, 1984), Teoc (Carpino et al, 1978), and Troc (Windholz and Johnston, 1967), have been used as protective groups for amino protection.

2. N-Sulfonyl derivatives Sulfonamide derivatives are frequently used in nitrogen heterocycles (Gribble et al, 1992), and arylsulfonyl (Fischer and Livschitz, 1915) groups are effective protective groups for a wide range of primary and secondary amines, but their deprotection requires drastic conditions. P-(Trimethylsilyl)ethanesulfonyl (Weinreb et al, 1986) derivatives are as stable as arylsulfonyl groups, but the cleavage step requires only gentle warming with TBAF or CsF.

3. N-Sulfenyl derivatives Sulfenamides are much more labile than sulfonamides, being sensitive to acids as well as to attack by nucleophiles. Their deprotection requires exceptionally mild conditions. Several sulfenyl groups are used for the protection of the amino function including tritylsulfenyl (Brandchaud, 1983), o-nitrophenylsulfenyl (Goerdeler and Holst, 1959), and pentachlorphenylsulfenyl (Kessler and Iselin, 1966).

WO 99/15510 PCT/AU98/00808 -4- 4. N-Alkyl derivatives Benzylamines give useful protection in reactions in which metal hydrides are used and the carbamates are not stable. Benzylamines are less susceptible to catalytic hydrogenolysis than benzyl ethers or benzyl esters, and thus selective deprotection can often be achieved (Goldstein et al, 1992). The trityl group (Sieber and Riniker, 1991) is used to protect amino acids, although its steric bulk and high acid lability is detrimental to peptide coupling. The 9-phenylfluorenyl (PhFl; Koskinen and Rapoport, 1989) group is used for the protection of primary and secondary amines. Its hydrophobicity, steric bulk and ease of introduction are similar to the trityl group, but the PhFl group is about 6000 times more stable to acid than the trityl group.

N-Silyl derivatives The high acid and moisture sensitivity of silylamines has been a major obstacle to their use in amino group protection. Butyldiphenylsilylamines (Overman and Okazaki, 1986) have remarkable stability towards strong basic conditions, but they are still very acid labile.

6. Imine derivatives The double bond of the imine function allows for the simultaneous protection of both N-H bonds of a primary amine. Imines are generally stable towards strongly basic conditions, but they are labile to aqueous acid. N-Silyl imines (Colvin et al, 1988), N-bis(methylthio)methyleneamines (Hoppe and Beckmann, 1979) and N-diphenylmethyleneamines (Polt et al, 1979) are valuable for the protection of amino groups in the synthesis of a-amino acids.

7. Enamine derivatives N-(5,5-Dimethyl-3-oxo-l-cyclohexenyl)amine (Halpern and James, 1964) is used to protect amino acids, giving vinylogous amide derivatives. These compounds can WO 99/15510 PCT/AU98/00808 be cleaved by treatment with either aqueous bromine or nitrous acid. The stability of the vinylogous amideprotected primary amines mainly depends on the structure of 1,3-dione and the functional group attached to the enamine double bond. The open chain N-(4-oxopent-2-enyl)-protected amines are labile towards aqueous and mildly acidic conditions. This acid sensitivity limits their use as synthetic reagents (Kellam, 1996). The cyclic 1,3diketone, 5,5-dimethylcyclohexane-l,3-dione (dimedone) reacts with dimethylformamide dimethylacetal affording dimethyl-2-(dimethylaminomethylene)cyclohexane-1,3-dione.

Bycroft et al (1993) used this reagent to synthesise Dmcprotected a-amino acids, and found remarkable stability towards acidic conditions. The deprotection of these compounds could be rapidly achieved by a dilute hydrazine solution at room temperature. The introduction of a methyl group to the enamine double bond provided the N-1-(4,4dimethyl-2,6-dioxocyclohexylidene)ethyl Dde-protective group, improving the stability towards secondary amines (Bycroft et al, 1993). The N-l-(4,4-dimethyl-2,6dioxocyclohexylidene)-3-methylbutyl-protected amino acids (Chan et al, 1995), carrying a bulkier group at the enamine double bond, had excellent base stability. N-l-(4-Nitro- 1,3-dioxoindan-2-ylidene)-ethyl (Nde; Kellam, 1996; Mosher and Meier, 1970) protection of amino acids gave similar vinylogous systems, and deprotection of these could be achieved in very mild conditions.

For many years chemists have attempted to transpose the solid-phase methodology which is routinely used for peptide synthesis to oligosaccharide synthesis, with varying degrees of success. The first attempt was approximately 25 years ago (Frechet and Schuerch, 1971; Frechet and Schuerch, 1972; Guthrie et al, 1971; Guthrie et al, 1973). However, the ozone-mediated deprotection product was an aldehyde-substituted glycoside. Danishefsky and coworkers described the solid phase synthesis of the Lewis b Antigen (Randolph et al, 1995) and N-linked WO 99/15510 PCT/AU98/00808 -6glycopeptides (Roberge et al, 1995) by initial attachment of the primary sugar unit of the oligosaccharide to a 1% divinylbenzene-styrene co-polymer support via a silyl ether linkage. The resin-bound sugar moiety was in this instance a glycal, with on-resin activation achieved via epoxidation of the double bond, and the resulting glycal residue acting as a sugar donor through nucleophile ring-opening of the epoxide. Since there are no colorimetric methods available to the sugar chemist to monitor on-resin glycosylations, the only means of assessing the progress of the reaction is by lysis of the oligosaccharide-resin bond and subsequent analysis of the cleavage product, usually by thin layer chromatography. The tetra-n-butylammonium fluoridemediated deprotection conditions required to cleave Danishefsky's silyl ether linker are both hazardous and slow. This, coupled with the requirement for on-resin activation of the tethered glycals, makes the overall strategy and methodology far from ideal.

In an alternative approach, Douglas and coworkers described the synthesis of D-mannopentose using a polyethyleneglycol w-monomethylether co-polymer and a succinoyl or an a,a'-dioxyxylyl diether linker (Douglas et al, 1995). The reactions were carried out in solution phase, with removal of unused reactants being achieved by precipitation of the oligosaccharide-polymer complex and subsequent washing. In the latter example, cleavage of the oligosaccharide-polymer bond was achieved through catalytic hydrogenation, which required exposure of the conjugate to 1 atm of H2 for 48 h to achieve respectable yields. This again is far too slow to allow effective monitoring-of individual glycosylation reactions. Yan et al reported sulphoxide-mediated glycosylation on a Merrifield resin, using a thiophenol linker for the attachment of the primary sugar residue (Yan et al, 1994). This method resulted in the construction of (1-6)-linked oligosaccharides, and was suitable for synthesis of both a- and 3-glycosidic linkages. However, the thioglycosidic linkage to the resin WO 99/15510 PCT/AU98/00808 -7dictates that similar sugar donors cannot be employed in this strategy.

Recently Rademann and Schmidt reported the use of trichloroacetimidate sugar donors to a resin bound sugar tethered via an alkyl thiol (Rademann and Schmidt, 1996); once again, however, this method precludes the use of the far superior thioglycoside sugar donors. Meanwhile, Adinolfi et al described the synthesis of disaccharides using a polyethyleneglycol-polystyrene resin, with connection of the first sugar to the polymeric support through a succinate spacer (Adinolfi et al, 1996).

However, the acid lability displayed by this linker means that the primary sugar cannot be linked to the resin via the glycosidic position.

These examples illustrate that the critical element in solid phase synthesis is the nature of the linker between the solid support and the initial synthon.

The linker must display excellent stability to the conditions of coupling and deprotection, yet in the case of solid phase oligosaccharide synthesis, it should also be rapidly and efficiently cleaved to allow monitoring of the progress of individual coupling reactions. The cleavage should ideally be achieved by the use of a relatively innocuous chemical reagent. There remains a need in the art for simple, efficient and economical methods for solidphase synthesis of oligosaccharides.

In our International Patent Application No. PCT/AU97/00544 (priority date 28 August 1996), we have shown several ways of immobilizing 2-acyl-5,5-dimethyl-l,3cyclohexanedione and of utilizing the immobilized compound in solid phase oligosaccharide synthesis. In our International Patent Application No. PCT/AU98/00131 (priority date 28 February 1997), we have shown that vinylogous amide protection of aminosugars could be achieved in simple reactions using Dde-OH and Nde-OH reagents. The entire disclosures of these specifications are incorporated herein by this reference. The Dde- and 8 Nde-protected monosaccharides survived most of the hydroxyl protective group manipulations and the reactions which occurred at the glycosidic center, affording a wide variety of sugar donors. These vinylogous amide-protected aminosugar donors were not neighbouring group active carbohydrates, giving anomeric mixtures of glycosides during the glycosylations. We have demonstrated the stability and the ease of deprotection of the Dde- and Nde-protected aminosugars in carbohydrate-based methodology.

Unfortunately even these protective strategies still present some difficulties.

The Dde-protected aminosugars are not stable in the presence of sodium cyanoborohydride and metal hydrides. These reagents are often used in benzylidene ring opening reactions and during benzyl protection of hydroxyl groups. This hydride sensitivity of the Dde group limits its application in carbohydrate chemistry.

The preparation of 2-acyl-dimedones is very often difficult. One of the major side reactions is Oacylation, which lowers the overall yields and causes difficult chromatographic purification problems.

Nde-protection of primary amines always gives a mixture of E/Z isomers which may not be separable, causing 25 difficult characterisation problems. The formation of 2acetyl-4-nitroindan-l,3-dione involves the reaction between 4-nitrophthalic anhydride and 2,4-pentanedione via a condensation and two rearrangements. This synthetic strategy does not give an opportunity to prepare Nde-OH 30 analogues.

We have now synthesized a family of novel compounds useful as protecting and linking groups for organic synthesis.

SUMMARY OF THE INVENTION In its most general aspect, the invention Sprovides a compound of general formula I \\melb fieslhome$\suzannet\Kcep\SpcA93303-98. I SPECIdoc 24/0502 ZV/IU ZUUZ 10:UJ rAAI 01 3 UZ4J0JJJ Uirr1iin nAUa i U D 9 R 0 N RI N R2 R O

I

in which each R is independently H; substituted or unsubstituted alkyl, aryl, alkenyl or alkynyl; or acyl; R' is hydrogen; an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloheteroaryl, cycloalkyl, heterocycloalkyl, alkanal, or thioalkanal group, each of which may be unsubstituted or substituted with substituents selected from amino, azido, halogen, hydroxyl, guanidino, carboxy, carboxylic acid ester, thio, carboxamide, alkylamino, dialkylamino, trialkylammonium, 10 and alkoxy; and when R' is hydrogen, then R 2 is a protected, unprotected or substituted sugar amino-; a glycosylamino-, or a glycosylamino group of an oligosaccharide; a mono- or oligosaccharide coupled 15 through a substituted or unsubstituted alkylamino-, arylamino-, cycloalkylamino, heteroalkylamino, heteroarylamino or heterocycloalkylamino group; an amino sugar; an amino acid or a peptide linked via a nitrogen atom; and 20 when R is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloheteroaryl, cycloalkyl, heterocycloalkyl, alkanal, or thioalkanal group, each of which may be substituted or unsubstituted then k 2 is a protected, unprotected or substituted sugar amino-; a glycosylamino-, or a glycosylamino group of an oligosaccharide; a mono- or oligosaccharide coupled through a substituted or unsubstituted alkylamino-, arylarnino-, cycloalkylamino, heteroalkylamino, heteroarylamino or heterocycloalkylamino group; an amino sugar; an amino acid or a peptide linked via a nitrogen atom; an 1IEnmnaU~mp peIJ333-f8.I PECduc 29110/02 I ZU/IU ZUU; iO;UJ rAA 01 J k4j54JJJ KirriinAAH aA WIJuo 10 alkylamino, dialkylamino, arylamino, or diarylamino group, each of which may be substituted or unsubstituted, with the provisos that a) when R 1 is unsubstituted phenyl; then R 2 is not unsubstituted aminophenyl (aniline) and b) when R 1 is methyl, then R 2 is not alkylamino substituted with a compound of formula I in which R is hydrogen and R 1 is methyl.

It will be clearly understood that in the general formulae of this specification, each of the substituent groups R, R 1 R1, and R 2 may itself be substituted, ie. one or more hydrogen atoms may be replaced by a substituent group.

For the purposes of this specification the term 15 "substituted" in the definitions of R, R 1 R1a and R 2 and in definitions of other substituents within this S. specification, means that the substituent is itself S. substituted with a group which modifies the general chemical characteristics of the chain. Preferred substituents include but are not limited to halogen, nitro, amino, azido, oxo, hydroxyl, thiol, carboxy, carboxy ester, carboxyamide, alkylamino, alkyldithio, alkylthio, alkoxy, acylamido, acyloxy, or acylthio, each of 1 to 3 carbon atoms. Such substituents can be used to 25 modify characteristics of the molecule as a whole, such as stability, solubility, and ability to form crystals. The person skilled in the art will be aware of other suitable substituents of similar size and charge characteristics which could be used as alternatives in a given situation.

Preferably each R has 1 to 6, more preferably 1 to 4 carbon atoms.

The compounds of the invention are useful in a wide variety of areas of organic chemistry. The compounds are especially useful in the solution and/or solid phase synthesis of oligosaccharides and peptides. Uses of the compounds of the invention thus include but are not limited to the following: n\saTannmlUcaSpSrp=ec3303-98.1 SPBiCLdoc 29102 iU/IU LUU Ia.;UJ rAA 01 a UI4.OJJi \ainrr±in UALY. W' u'I 11 1. Linker groups for solid-phase oligosaccharide synthesis; 2. N-protecting groups for protection of amino sugars in oligosaccharide synthesis; 3. Linker groups for solid phase organic synthesis; 4. N-protecting groups for organic synthesis; N-side chain and/or Na protecting groups for solid or solution phase peptide synthesis; 6. Amino protecting groups for sugars, peptides and organic compounds, affording an additional free enamine; 7. Certain compounds of the invention are chiral; these are useful in resolution of enantiomers and .*oo 15 in stereospecific synthesis.

8. Linker groups for coupling of a starter group to a resin for solid phase synthesis of oligosaccharides, peptides and other organic compounds.

Thus in a second aspect, the invention provides an N-protecting group for oligosaccharides, amino acids, peptides or organic compounds.

An example of the application of this group for the protection of amino groups during oligosaccharide synthesis is shown in general formula I defined above in 25 which R 2 is an amino sugar; a glycosylamino-, or a glycosylamino group of an oligosaccharide; a mono- or oligosaccharide coupled through a substituted or unsubstituted alkylamino-, arylamino-, cycloalkylamino, heteroalkylamino, heteroarylamino or heterocycloalkylamino group; or an amino acid or a peptide linked via a nitrogen atom.

Preferably R 2 is a protected, unprotected or substituted amino sugar, a glycosylamino-, or a glycosylamino group of an oligosaccharide.

Alternatively, R 2 is an oligosaccharide-O-CH2- RA& (C6H 4 monosaccharide-0-CH 2 -(C6H4)-NH-, x oligosaccharide-C02CH2-(C6H 4 or monosaccharide- Hn.44uuxrampSpocM3Il.9 I SPCoA 2W1IO 29/10 2002 15:03 FAX 61 3 92438333 GRIFFITH HACK 1008 12 C02CH2-(C6H4)-NH group.

In a third aspect the invention provides a support of general formula VI for solid-phase synthesis of oligosaccharides, peptides or organic compounds, comprising a resin and a linker covalently attached to the resin: N- R'a-RESIN N A R 0

VI

9 in which R 2 is as defined above, and 10 Rla is a substituted or unsubstituted alkyl, cycloalkyl, heteroalkyl, heteroaryl, heterocycloalkyl or carboxylamido spacer group which is directly coupled to the resin, or which may optionally be coupled to the resin via a suitable covalent linkage, which is stable to 15 conditions of oligosaccharide synthesis and cleavage, and

R

2 is a protected, unprotected or substituted sugar amino-; a glycosylamino-, or a glycosylamino group 0.000: of an oligosaccharide; a mono- or oligosaccharide coupled 20through a substituted or unsubstituted alkylamino-, arylamino-, cycloalkylamino, heteroalkylamino,

S

heteroarylamino or heterocycloalkylamino group; an amino sugar; an amino acid or a peptide linked via nitrogen atom; an alkylamino, dialkylamino, arylamino, or diarylamino group, each of which may be substituted or unsubstituted; OH, O'M, O-alkyl, O-acyl, 0-aryl, alkylthio, S-aryl, acylthio, alkylsulfonyl or arylsulfonyl, each of which may be substituted or unsubstituted; and M is a metal ion, or an organic or inorganic cation.

A wide variety of suitable cations is known in the art. The metal ion can be mono- or multivalent, and Smay form a complex salt.

RSatwc\KCc\SsPC4AQ33o3-9t1 SPWdc~c 29/1002 I ZU/lU iUUL 10.;U rAA 01 0 UL4OOJJJ Kimrril nAAlf IL-j UUo 13 The covalent linkage may suitably be provided by a -CONH-, -COO-, -COS-, -CH=N-, -NHCONH-, -NHCSNH, -NHNH- grouping, eg. Spacer-CONHresin, Spacer-O-resin, Spacer-S-resin, Spacer-S-S-resin, Spacer-CO2-resin, Spacer-CH=N-resin, Spacer-NHCONH-resin, Spacer-NHCSNH-resin, Spacer-NHNH-resin. Other possible covalent linking groups will be known to those skilled in the art.

In a particularly preferred embodiment, the linker is of general formula VI R 0 N Rn-Resin N R R 0

VI

in which R is as defined in formula I above, in which a compound of general formula II is 15 directly coupled to the resin support, or may optionally be coupled to the resin support via a suitable covalent linkage which is stable to conditions of oligosaccharide 0 synthesis and cleavage.

The covalent linkage may suitably be provided by 20 a -CONH-, -COO-, -COS-, -CH=N-, -NHCONH-, -NHCSNH, or -NHNH- grouping, eg. Spacer-CONHresin, Spacer-O-resin, Spacer-S-resin, Spacer-S-S-resin, Spacer-C02-resin, Spacer-CH=N-resin, Spacer-NHCONH-resin, Spacer-NHCSNH-resin, Spacer-NHNH-resin. Other possible covalent linking groups will be known to those skilled in the art.

The resin may be any resin which swells in water and/or in an organic solvent, and which comprises one of H:umnaUucl\xpSpet3.V1 .SPFXsPCItoc 29/10/02 29/10 2002 15:04 FAX 61 3 92438333 GRIFFITH HACK [a 14 THIS PAGE IS INTENTIONALLY BLANK 0 0000 0 000009 00 ILtannat.EapiSpedfl23-utii spECI,doc 2911o/0y2 fVlU ZUUZ 10:u4 YAA& 01 J Ui:4JOJJJ tiX1ltI JAUah li~ U I I 15 THIS PAGE IS INTENTIONALLY ]BLANK S. @0 0 00000 0 @9 @0 @0 0 @000 0@0000 0* 0 @0 9 0 @0 R~summmxeeplspcrtyno.13-pa.1 SPEfldoe 1-9/1M2 WO 99/15510 PCT/AU98/00808 -16the following substituents: halogen, hydroxy, carboxyl, SH,

NH

2 formyl, SO 2

NH

2 or NHNH 2 for example methylbenzhydrylamine (MBHA) resin, amino or carboxy tentagel resins, or 4-sulphamylbenzyl AM resin. Other suitable resins will be known to those skilled in the art.

Alternatively, supports such as controlled-pore glass or soluble polymer supports may be used. These are well known in the art.

The invention also provides a method of solidphase synthesis of oligosaccharides, comprising the step of sequentially linking mono- or oligosaccharide groups to a support as described above.

The linker may be synthesised directly on the resin in a stepwise manner prior to the coupling of the initial sugar group, or the linker-initial sugar conjugate may be synthesised in solution phase and subsequently coupled to the solid support, with subsequent sugars being sequentially attached. Preferably the second and all subsequent sugar groups are coupled to the oligosaccharide chain-resin conjugate after the last sugar in the oligosaccharide chain is partially deprotected.

The first sugars attached to the resin-linker unit may be unprotected, partially protected or fully protected glycosides, aminoglycosides, or ether- or amino-linked sugars.

Preferably the first sugar coupled to the resin is an aminosugar, an aminoglycoside or an aminooligosaccharide, or a glycosyl amines of an oligosaccharide.

In one particularly preferred embodiment the support comprises a resin, a linker and a saccharide selected from the group consisting of monosaccharide, oligosaccharides, or aminosaccharides and aminooligosaccharides.

The building block mono- or oligosaccharidedonors may be any activated sugar, including but not limited to orthoesters, thio-orthoesters, cyanoalkylidene WO 99/15510 PCT/AU98/00808 -17derivatives, l-O-acyl sugars, amino sugars, acetimidates, trichloroacetimidates, thioglycosides, aminoglycosides, aminoligosaccharides, glycosylamines of oligosaccharides, glycosyl thiocyanates, pentenyl glycosides, pentenoylglycosides, isopropenyl glycosides, glycals, tetramethylphosphoro diamidates, sugar diazirines, selenoglycosides, phosphorodithioates, glycosyldialkylphosphites, glycosylsulphoxides and glycosylfluorides.

Preferably partial sugar deprotection is achieved by using acyl-type, trityl, methoxytrityl, methoxybenzyl, various silyl and/or photolabile protecting groups in addition to permanent ether-type protecting groups. This permits the synthesis of branched oligosaccharides by using two orthogonal hydroxy-protecting groups on a single sugar donor.

The synthesised oligosaccharide can be cleaved from the resin using ammonia, hydrazine or a primary amine, such as butylamine or cyclohexylamine. For the preparation of aminoglycosides, ammonia or a suitable primary amine in an organic solvent is preferably employed. For the preparation of hydrazides, hydrazine in water or an organic solvent is preferably employed. For the preparation of oligosaccharides, ammonia in water or organic solvent is preferably employed, followed by acidification. When the linker contains a 4-aminobenzyl moiety, after cleavage as described above the first sugar is released still protected by the aminobenzyl group; this can be removed by hydrogenation if desired.

In a preferred embodiment, the invention provides a reagent for solution phase synthesis of sugar-containing compounds, comprising a barbituric acid derivative compound of general formula II as defined above.

The compounds of the invention are suitable for use as protecting groups in methods of solid-phase oligosaccharide synthesis, in which sugar units are linked to a resin. Any suitable linker compound may be used, WO 99/15510 PCT/AU98/00808 -18including compounds of the invention. It is contemplated that linkers and methods described in our earlier application, PCT/AU97/00544, are also suitable for use with the compounds of this invention.

Thus in a fourth aspect the invention provides a linker-saccharide complex, comprising a linker group and a starting compound comprising a protecting group of general formula I or II as defined above. Any suitable linker may be used, including the compounds of the invention. Again, it is contemplated that linkers and methods described in PCT/AU95/00544 may be used.

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 solution phase method of the invention may, for example, be used for combinatorial synthesis of aminoglycoside compounds.

The invention also provides kits useful in solution phase synthesis or combinatorial synthesis of oligosaccharides or peptides, comprising either a) a resin-linker-saccharide or resin-linkerpeptide (or amino acid) support, b) a linker-saccharide or linker-peptide (or amino acid) complex, or c) a resin-linker support, according to the invention, as described above.

For peptide synthesis it may be convenient in some circumstances to start with a resin-linker-amino acid support or linker-amino acid complex, while in others a starter peptide may more suitably be provided in the support or linker complex. The kit may optionally also comprise one or more further reagents such as protecting agents, deprotecting agents, and/or solvents suitable for solid phase or combinatorial synthesis. The person skilled in the art will be aware of suitable further reagents.

WO 99/15510 PCT/AU98/00808 -19- Different types of kit can then be chosen according to the desired use.

The invention also provides a kit useful in solid phase synthesis or combinatorial synthesis of oligosaccharides, comprising a linker-saccharide complex according to the invention, as described above. The kit may optionally also comprise one or more further reagents such as protecting agents, deprotecting agents, 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 Abbreviations used herein are as follows: Ac AcOH

ADA

Alloc Boc Bu Cbz

DBU

DCC

Dde

DMAP

Dmc

DMF

EtOH FAB MS Fmoc Acetyl Acetic acid 5-Acyl-l,3-dimethylbarbituric acid Allyloxycarbonyl tert-Butoxycarbonyl butyl Benzyloxycarbonyl 1,8-Diazabicyclo[5.4.0]undec-7-ene N,N'-Dicyclohexylcarbodiimide N-l-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-ethyl 4-Dimethylaminopyridine N-(4,4-Dimethyl-2,6-dioxocyclohexylidenemethylene) N,N'-Dimethylformamide Ethanol Fast atom bombartment mass spectrometry 9-Fluorenylmethoxycarbonyl WO 99/15510 PCT/AU98/00808

MBHA

Me MeOH Nde

NMR

ODmab PhF1

TBAF

TEAB

Teoc

TNBS

Troc methylbenzylhydramine Methyl Methanol l-(4-Nitro-l,3-dioxoindan-2-ylidene)ethyl Nuclear magnetic resonance -{N-[1-(4,4-dimethyl-2,6-dioxocyclohexyl-idene)- 3-methylbutyl]-amino}benzyl alcohol 9-Phenylfluorenyl Tetrabutylammonium fluoride Tetraethylammonium bromide 2-(Trimethylsilyl)ethoxycarbonyl 2,4,6-trinitrobenzene sulphonic acid 2,2,2-Trichloroethoxycarbonyl 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 and structures.

26/08 2002 15:52 FAX 61 3 92438333GRFIH AC 103 GRIFFITH HACK [a 013 21 Table 1 Compounds 1-20

N.

CH3 Compound R 2

R

1 1 OH

CH

3 2 NHBu

CH

3 3 OHi Ph 4 NHBu Ph OH 9-f luorenyl 6 OH

CH

2 C1 7 OH CHC1 2 8 OH Bn 9 OH CHPh 2 10 OH -(CH 2 3

COOH

11 OH t-Bu 12 OH I-adamantyl 13

NH

2 CC1 3 14 -NHCH 2 COOll CH 3 i5 -NHCH 2 COOH Ph 16 -NHCH 2 COOH Bn 17 -NHOH Ph 1.8 -NI{NHCOCH 3 Ph 2.9

-NH-NIH

2 Ph 202 Ph SFECI.Joc 26,()PA)2 WO 99/15510 WO 9915510PCT/AU98/00808 22 Table 2 Compounds 21-29 Compound R 21 CH- 3 22 Ph 23 9-f luorenyl 24 Bn CH-Ph 2 26 (CH 2 3

COOH

27 NH 2 28 t-Bu 129 1-adarnantyl wo 99/15510 WO 9915510PCT/AU98/00808 23 N-Me me 0 32 R =Ac 37 R =H HO

OH

0 (CH 2 3 0 Me,-N 0 N-Me 0 e Me 0 33 35 R =NH 2 36 R =OH WO 99/15510 PCT/AU98/00808 -24- 0 OH Q HO 0

CH

3 HO N SMe

NH

0 MeN Me 0 38 39 We have now developed a novel enamine-type protective system, including the preparation of reagents, and methods for selective amino group protection and deprotection. This has been illustrated by synthesizing a number of 5-acyl-l,3-dimethyl-barbituric acids (ADA) (Examples 1-11). During the syntheses only C-acyl products were formed; no O-acylation was observed. The of 1,3-dimethylbarbituric acid was successfully carried out using carboxylic acids in the presence of DCC and DMAP (Examples 5 to The more reactive acyl chlorides (Examples 3 to 4) and anhydrides (Examples 1 to 2) were also used, giving the same products in a DMAP-catalyzed reaction. Trichloroacetonitrile was used to construct a similar structure in the present of DBU (Example The 5-acyl-l,3-dimethylbarbituric acids were easily crystallized from polar solvents, avoiding the need for chromatographic purifications. These reagents are very cheap and easy to synthesize in a single reaction from the readily available 1,3-dimethyl-barbituric acid. We have used the 5-acyl-l,3-dimethylbarbituric acid reagents to prepare a wide variety of protected primary alkylamines (Examples 12-13), aminosugars (Examples 22 to 28) and amino acids (Examples 14 to 16).

WO 99/15510 PCT/AU98/00808 The ADA-protected aminosugars can be used as aminosugar acceptors and aminosugar donors for solid or solution phase oligosaccharide synthesis. The ADAprotected amino acids are particularly useful as reagents for solid-phase peptide and glycopeptide syntheses, because they are unable to form oxazolones during the coupling reactions. Thus, no racemization can occur during the peptide bond formation (racemization can only occur in base-catalyzed proton abstraction). The ADA-protection is ideally orthogonal to the Boc-protection and quasiorthogonal to the Fmoc system.

We have demonstrated that the system can be used for the protection of hydroxylamines (Example 17), hydrazines (Example 19) and hydrazides (Example 18). The vinylogous amide protection of amino groups was efficiently achieved by simply refluxing the unprotected amines with the precursor (5-acyl-l,3-dimethylbarbituric acid) in abs.

EtOH.

The ADA-protected derivatives are very stable in a wide range of reactions and work-up conditions.

Different reagents (NH 3

N

2

H

4 NH20H, n-BuNH 2 BnNH 2

NH-

NHCOCH

3

N

2

H

4 xAcOH, NaOH) have been developed for the cleavage of the protecting groups (Examples 17 to 20). The speed of protection and cleavage depends on the electronic and steric effects of the 5-acyl functional group.

We have also synthesized bifunctional 5-acyl-l,3dimethylbarbituric acids (Example 11), which can be used as linkers for solid phase organic chemistry. We have successfully immobilized a bifunctional 5-acyl-l,3dimethylbarbituric acid producing a "resin-linker conjugate" (Example 35). We have proved that this "resinlinker conjugate" was suitable for solid phase oligosaccharide synthesis by immobilizing a monosaccharide (Example 32), deprotecting its hydroxyl groups (Example 33) and later realising it during the cleavage (Example 33).

We have demonstrated that the resin-linker conjugate was reusable, regenerating the original hydroxyl function with WO 99/15510 PCT/AU98/00808 -26aqueous base treatment (Example 36). Alternatively the "amino-substituted resin-linker conjugate" itself may be used for the next immobilization (Example 34).

The introduction of another reactive centre into the protecting group makes the system more flexible. -Using 5-chloroacetyl-l,3-dimethylbarbituric acid, we have synthesised a chiral carbohydrate containing reagent (Example 31) for protection of organic compounds bearing an amino functionality. These types of molecules are especially suitable for resolution of enantiomers.

The 5-trichloroacetimino-l,3-dimethyl-barbituric acid gave rare 1,1-elimination in the reaction with primary amines, affording a novel type of compound (Example 29).

Example 1 5-Acetyl-l,3-Dimethyl-2,4,6(1H,3H,5H)- Pyrimidinetrione (Dtpc-OH) 1 A mixture of 1,3-dimethylbarbituric acid (10 g, 64.04 mmol), 4-dimethylaminopyridine (9.49 g, 158.0 mmol) in dry CH2C1 2 (190 ml) was cooled to 0°C and acetic anhydride (7.35 ml, 77.9 mmol) added dropwise in 15 min.

The reaction mixture was stirred overnight at room temperature, diluted with CH 2 C12 (500 ml) and washed with 2 N HC1 solution (80 ml). The organic phase was dried over MgS0 4 and evaporated. The residue was crystallised from MeOH, giving 5-acetyl-l,3-dimethyl-2,4,6(1H,3H,5H)pyrimidinetrione 1 (8.6 g, 68%).

Rf 0.37 (EtOAc/hexane 2:1); FAB MS C 8

H

10

N

2 0 4 (198.18) m/z 199 (100), 183 (18) H NMR (CDC13) d 17.26 1H, OH), 3.36, 3.32 (2s, 6H, 2 NCH 3 2.71 3H, CH 3 Example 2 5-Chloroacetyl-l,3-Dimethyl-2,4,5(1H,3H,5H)- Pyrimidinetrione (Dtpc-OH) 6 A mixture of 1,3-dimethylbarbituric acid (5.00 g, 32.02 mmol), 4-dimethylaminopyridine (9.76 g, 80.05 mmol) in dry CH 2 C12 (75 ml) was cooled to 0 0 C and chloroacetic WO 99/15510 PCT/AU98/00808 -27anhydride (6.57 g, 38.46 mmol) added. The reaction mixture was stirred at room temperature overnight, diluted with

CH

2 C1 2 (150 ml) and washed with 2 N HC1 solution (40 ml).

The organic phase was dried over MgS0 4 and evaporated. The residue was crystallised from MeOH, giving 1,3-dimethyl-2,4,6(1H,3H,5H)-pyrimidinetrione 6 (4.57 g, 61%).

Rf 0.41 (hexane/EtOAc/AcOH 10:5:0.1); FAB MS C 8

H

9 C1N 2 0 4 (232.62) m/z 233 (100), 197 183 H NMR (CDC1 3 d 17.93 1H, OH), 4.97 2H, CH 2 3.41, 3.34 (2s, 6H, 2 NCH 3 Example 3 5-Benzoyl-l,3-Dimethyl-2,4,6(1H,3H,5H)- Pyrimidinetrione (Dtpb-OH) 3 A mixture of 1,3-dimethylbarbituric acid (5 g, 32.02 mmol), 4-dimethylaminopyridine (4.74 g, 38.79 mmol) in dry CH 2 C1 2 (75 ml) was cooled to 0°C and benzoyl chloride (4.95 g, 35.22 mmol) added dropwise in 15 min.

The reaction mixture was stirred for 3 h at room temperature, diluted with CH 2 C12 (150 ml) and washed with 2 N HC1 solution (40 ml). The organic phase was dried over MgSO 4 and evaporated. The residue was crystallised from diisopropylether then recrystallised from MeOH, giving 5-benzoyl-l,3-dimethyl-2,4,6(lH,3H,5H)-pyrimidinetrione 3 (5.32 g, 64%).

Rf 0.45 (EtOAc/hexane/TFA 10:15:0.1); FAB MS C1 3

H

12

N

2 0 4 (260.25) m/z 283 [M+Na] 261 (100), 245 183 IH NMR (CDC1 3 d 16.58 1H, OH), 7.57 7.45 Ar-H), 3.44, 3.27 (2s, 6H, 2 NCH 3 WO 99/15510 PCT/AU98/00808 -28- Example 4 5-Pivaloyl-1,3-dimethyl-2,4,6(1H,3H,5H)pyrimidinetrione (Dtppe-OH) 11 A mixture of 1,3-dimethylbarbituric acid (5 g, 32.02 mmol), 4-dimethylaminopyridine (4.69 g, 38.42 mmol) in dry CH 2 C12 (75 ml) was cooled to 0°C and pivaloyl chloride (4.24 g, 35.22 mmol) added dropwise in 15 min.

The reaction mixture was stirred at room temperature overnight, diluted with CH 2 C12 (150 ml) and washed with 2 N HC1 solution (40 ml). The organic phase was dried over MgSO 4 and evaporated. The residue was purified by chromatography using hexane/EtOAc/AcOH 15:5:0.1 as the mobile phase to give 5-pivaloyl-1,3-dimethyl- 2,4,6(1H,3H,5H)-pyrimidinetrione 11 (5.46 g, 71%).

Rf 0.65 (hexane/EtOAc/AcOH 15:5:0.1); FAB MS C 11

H

16

N

2 0 4 (240.26) m/z 263 [M+Na] 241 [M+H] (100), 223 183 1H NMR (CDC1 3 d 19.14 1H, OH), 3.38, 3.33 (2s, 6H, 2 NCH 3 1.41 9H, 3 CH 3 Example 5 5-(9-Fluorenylcarbonyl)-1,3-Dimethyl- 2,4,6(1H,3H,5H)-Pyrimidinetrione (Dtpf-OH) A mixture of 1,3-dimethylbarbituric acid (2.5 g, 16.01 mmol), 9-fluorenylcarboxylic acid (5.05 g, 24.01 mmol), 4-dimethylaminopyridine (0.98 g, 8.00 mmol) in dry CH 2 C1 2 (15 ml) was cooled to 0°C and 1,3dicyclohexylcarbodiimide (3.30 g, 16.01 mmol) added. The reaction mixture was stirred at room temperature overnight and filtered. The solid was washed with CH 2 C12 (50 ml) and the combined solution was washed with 2 N HC1 solution ml). The organic phase was dried over MgSO 4 and evaporated. The residue was crystallised from recrystallised from MeOH giving 5-(9-fluorenyl-carbonyl)- 1,3-dimethyl-2,4,6(1H,3H,5H)-pyrimidinetrione 5 (2.85 g, 69%).

Rf 0.49 (EtOAc/hexane/TFA 10:25:0.1); WO 99/15510 PCT/AU98/00808 -29- FAB MS C 2 oH1 6

N

2 0 4 (348.35) m/z 349 (100), 338 183 164 (71).

H NMR (CDC1 3 d 17.33 1H, OH), 7.81 2H, 2 Ar-H), 7.42 4H, 4 Ar-H), 7.30 2H, 2 Ar-H), 6.92 1H, CH), 3.48, 3.40 (2s, 6H, 2 NCH 3 Example 6 5-Dichloroacetyl-l,3-Dimethyl- 2,4,6(1H,3H,5H)-Pyrimidinetrione (Dtpd-OH) 7 A mixture of 1,3-dimethylbarbituric acid (5.00 g, 32.05 mmol), dichloroacetic acid (6.19 g, 48.03 mmol), 4-dimethylaminopyridine (1.95 g, 16.01 mmol) in dry CH 2 C1 2 ml) was cooled to 0°C and 1,3-dicyclohexylcarbodiimide (7.26 g, 35.22 mmol) added. The reaction mixture was stirred at room temperature overnight and filtered. The solid was washed with CH 2 C1 2 (150 ml) and the combined solution was washed with 2 N HC1 solution (40 ml). The organic phase was dried over MgSO 4 and evaporated. The residue was crystallised from MeOH giving 1,3-dimethyl-2,4,6(lH,3H,5H)-pyrimidinetrione 7 (5.41 g, 63%).

Rf 0.27 (hexane/EtOAc/AcOH 10:5:0.1); FAB MS CsH 8 Cl 2

N

2 0 4 (267.07) m/z 289 267 (100), 231 197 183 (24).

H NMR (CDC1 3 d 17.94 1H, OH), 7.91 1H, CH), 3.43, 3.35 (2s, 6H, 2 NCH 3 Example 7 5-Phenylacetyl-l,3-Dimethyl-2,4,6(1HH,35H)- Pyrimidinetrione (Dtpp-OH) 8 A mixture of 1,3-dimethylbarbituric acid (5.00 g, 32.05 mmol), phenylacetic acid (6.53 g, 48.03 mmol), 4-dimethylaminopyridine (1.95 g, 16.01 mmol) in dry CH 2 C12 ml) was cooled to 0 C and 1,3-dicyclohexylcarbodiimide (7.26 g, 35.22 mmol) added. The reaction mixture was stirred at room temperature overnight and filtered. The solid was washed with CH 2 C12 (150 ml) and the combined solution was washed with 2 N HC1 solution (40 ml). The WO 99/15510 PCT/AU98/00808 organic phase was dried over MgSO 4 and evaporated. The residue was crystallized from MeOH giving 1,3-dimethyl-2,4,6(1H,3H,5H)-pyrimidinetrione 8 (6.10 g, 69%).

Rf 0.41 (hexane/EtOAc/AcOH 10:5:0.1); FAB MS C1 4 Hi 4

N

2 0 4 (274.27) m/z 297 [M+Na] 275 (100), 257 183 (31).

1H NMR (CDC1 3 d 17.61 1H, OH), 7.54 7.26 5 Ar-H), 4.49 2H, CH 2 Ar), 3.38, 3.34 (2s, 6H, 2 NCH 3 Example 8 5-Diphenylacetyl-l,3-dimethyl- 2,4,6(1H,3H,5H)-pyrimidinetrione (Dtpd-OH) 9 A mixture of 1,3-dimethylbarbituric acid (5.00 g, 32.05 mmol), diphenylacetic acid (10.19 g, 48.03 mmol), 4-dimethylaminopyridine (1.95 g, 16.01 mmol) in dry CH 2 C1 2 ml) was cooled to 0°C and 1,3-dicyclohexylcarbodiimide (7.26 g, 35.22 mmol) added. The reaction mixture was stirred at room temperature overnight and filtered. The solid was washed with CH 2 C12 (150 ml) and the combined solution was washed with 2 N HC1 solution (40 ml). The organic phase was dried over MgS04 and evaporated. The residue was crystallized from EtOH giving 1,3-dimethyl-2,4,6(1H,3H,5H)-pyrimidinetrione 9 (6.70 g, 59%).

Rf 0.64 (hexane/EtOAc/AcOH 10:5:0.1); FAB MS C 2 0H 8 iN 2 0 4 (350.36) m/z 373 351 (100), 338 333 (16).

1H NMR (CDC13) d 18.28 1H, OH), 7.32 7.27 Ar-H), 7.02 1H, CHAr 2 3.36, 3.31 (2s, 6H, 2 NCH 3 Example 9 5-(1-Adamantanecarbonyl)-1,3-Dimethyl- 2,4,6(1H,3H,5H)-Pyrimidinetrione (Dtpa-OH) 12 A mixture of 1,3-dimethylbarbituric acid (5.00 g, 32.05 mmol), 1-adamantanecarboxylic acid (8.65 g, 48.03 mmol), 4-dimethylaminopyridine (1.95 g, 16.01 mmol) WO 99/15510 PCT/AU98/00808 -31in dry CH 2 C1 2 (30 ml) was cooled to 0°C and 1,3-dicyclohexylcarbodiimide (7.26 g, 35.22 mmol) added.

The reaction mixture was stirred at room temperature overnight and filtered. The solid was washed with CH 2 C12 (150 ml) and the combined solution was washed with 2 N HC1 solution (40 ml). The organic phase was dried over MgS04 and evaporated. The residue was crystallized from MeOH giving 5-(1-adamantanecarbonyl)-1,3-dimethyl- 2,4,6(1H,3H,5H)-pyrimidinetrione 12 (7.10 g, 69%).

Rf 0.57 (hexane/EtOAc/AcOH 15:5:0.1); FAB MS C1 7

H

22

N

2 0 4 (318.37) m/z 319 (100), 301 223 183 (94).

1H NMR (CDC1 3 d 19.23 1H, OH), 3.38, 3.35 (2s, 6H, 2 NCH 3 2.18, 2.07 (2s, 12H, 6 CH 2 1.79 3H, 3 CH).

Example 10 5-Trichloroacetimino-l,3-dimethyl- 2,4,6(1H,3H,5H)-pyrimidinetrione (Dtpe-NH 2 13 A mixture of 1,3-dimethylbarbituric acid (5.00 g, 32.02 mmol), 4-dimethylaminopyridine (1.95 g, 16.01 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene /DBU/ (10 drops) in dry

CH

2 C1 2 (50 ml) was cooled to 0°C and trichloroacetonitrile (13.87 g, 96.06 mmol) added dropwise in 15 min. The reaction mixture was stirred at 0°C for 30 min then at room temperature for 3 h, diluted with CH 2 C12 (50 ml) and washed with 1 N KHS0 4 solution (10 ml). The organic phase was dried over MgSO 4 and evaporated. The residue was crystallized from MeOH giving 5-acetimino-1,3-dimethyl- 2,4,6(1H,3H,5H)-pyrimidinetrione 13 (6.22 g, Rf 0.61 (EtOAc/hexane 1:1); FAB MS C 8

H

8 Cl 3

N

3 0 3 (300.53) m/z 322 300 (100), 264 243 207 183 (17).

'H NMR (CDC1 3 d 13.13, 7.83 (2s, 2H, 2 NH), 3.37, 3.33 (2s, 6H, 2 NCH 3 WO 99/15510 WO 99/ 5510PCT/AU98/00808 32 Example 11 5- (4-Carboxybutyryl) 3-Dimethyl- 2,4, 6(lH,3H,5H)-Pyrimidinetrione (Dtpp-OH) and l,5-bis-(1,3-Dimethyl-2,4,6-(lH,3H,5H)- Pentane 33 A mixture of 1,3-dimethylbarbituric acid (5.00 g, 32.02 mnol), 4-dimethylaminopyridine (9.789 g, 80.05 mmol) in dry CH 2 C1 2 (75 ml) was cooled to 0 0 C and glutaric anhydride (4.38 g, 38.42 mmol) added. The reaction mixture was stirred overnight at room temperature, diluted with

CH

2 Cl 2 (150 ml) and washed with 2 N HCl solution (40 ml) The organic phase was dried over MgSO 4 and evaporated. The residue was crystallized from AcOH giving 1,5-bis-(l,3dimethyl-2, 4,6 (lH, 3H, 5H) -trioxopyrimidin-5-ylidene) dihydroxy pentane 33 (1.2 g).

Rf 0. 71 (CH 2 Cl 2 /MeOH/AcOH 96:3: 1); FAB MS C 1 7

H

2 oN 4 0 8 (408.36) m/z 431 409 (100).

1H NMR (CDCl 3 d 17. 67 2H, 2 OH) 3 .37, 3.31 (2s, 12H, 4 NCH 3 3 .27 4H, 2 CH 2 2. 12 (in, 2H, CH 2 The filtrate was evaporated and the residue was crystallized from toluene to give 5-(4--carboxybutyryl)-l,3dimethyl-2,4,6(lH,3H,5H)-pyrimidinetrione 10 (2.10 g, 24%) Rf 0. 66 (CH 2 C1 2 /MeOH/AcOH 96: 3:1) FAB MS C11H1 4

N

2 0 6 (270.24) m/z 293 (10) 271 (100) 253 (76) 225 (22) 211 (20) 'H NMR (CDCl 3 d 17.67 1H, OH) 3.37, 3.32 (2s, 6H, 2 NCH 3 3.23 2H, CH 2 2.48 2H, CH 2 2.05 (in, 2H, Gil 2 WO 99/15510 WO 99/ 5510PCT/AU98/00808 33 Example 12 N-[1-(1,3--dimethyl-2,4,6(lH,3H,5H)ethyl] 1-butylamine 2 5-Acetyl-1, 3-dimethyl-2, 4,6 (1H, 3H, 5H) pyrimidinetrione (100 mg, 0.50 mmol) was dissolved inn-butylamine (10 ml) and stirred at room temperature overnight. The solvent was evaporated, the residue was washed with ether to give N-[1-(l,3-dimethyl- 2,4,6 (lH, 3H, 5H) -trioxopyrimidin--5-ylidene) ethyl] 1-butylarnine 2 (121 mg, Rf 0.33 (EtOAc/hexane 2:1); FAB MS C1 2

H,

9

N

3 0 3 (253 .28) m/z 266 [M+Na] 254 (100) 195 (14) 'H NMR (CDCl 3 d 12.5S5 1H, NH) 3. 44 (in, 2H, CH 2 3. 31, 3.30 (2s, 6H, 2 NCH 3 2.68 3H, CH 3 1.69, 1.45 (2m, 4H, 2 CH 2 0.97 3H, CH 3 Example 13 N-[l-(l,3-dimethyl-2,4,6(lH,3H,5H)trioxopyrimidin-5-ylidene) phenylmethylI 1-butylamine 4 A mixture of 5-benzoyl-l,3-dimethyl- 2,4,6(lH,3H,5H)-pyrimidinetrione (500 mng, 1.92 nunol) and N,N-diisopropylethylamine (248 mg, 1.92 minol) in n-butylamine (10 ml) was ref luxed for 2 hours. The solvent was evaporated, the residue was washed 1 M KHS0 4 solution, dried and evaporated. The residue was washed with ether to give N-[l-(l,3-dimethyl-2,4,6(lH,3H,5H)-trioxopyrimidin-5ylidene)phenylmethyl] 1-butylamine 4 (575 mng, Rf 0.41 (EtOAc/hexane/TFA 10:15:0.1); FAB MS C1 7

H

2 1

N

3 0 3 (315.36) m/z 338 [M+NalY (16) 316 1M+H] (100) 307 (14) 1H NM~R (CDCl 3 d 12.42 1H, NH) 7.48 (mn, 3H, 3 Ar-H) 7. 17 (in, 2H, 2 Ar-H) 3.37, 3 .15 (2s, 6H, 2 NCH 3 3. 04 (in, 2H, CH 2 1.52, 1.32 (2mn, 4H, 2 CH 2 0.86 3H, CHA WO 99/15510 WO 9915510PCT/AU98/00808 34 Example 14 N-[l-(l,3-dimethyl-2,4,6(lH,3H,5H)ethyll glycine 14 A mixture of 5-acetyl-l,3-dimethyl- 2,4,6(lH,3H,5H)-pyrimidinetrione (396 mg, 2.00 mmol), glycine (100 mg, 1.33 mmol) and N,N-diisopropyl-ethylamine (172 mg, 1.33 mmol) in abs. EtOH (10 ml) was stirred under ref lux overnight. The solvent was evaporated, the residue was taken up in CH 2 Cl 2 (100 ml) washed with 1 M KHS0 4 solution (10 ml) The resulting suspension was filtered, the precipitate was washed with ether and recrystallized from EtCH giving N-[1-(l,3-dimethyl-2,4,6(lH,3H,5H)glycine 14 (290 mg, R, 0. 28 (CH 2 Cl 2 /EtOAc /MeOH 10: 7:1) FAB MS Cj 0

H

1 3

N

3 0 5 (255.22) m/z M% 278 256 (100), 210 (44).

IH NIMR (CDCl 3 d 12.58 1H, NH) 3. 64 2H, CH 2 3.34, 3.31 (2s, 6H, 2 NCH 3 2. 69 3H, CH- 3 Example 15 N-[l-(l,3-dimethyl-2,4,6(lH,3H,5H)trioxopyrimidin- 5-yl idene) phenylmethyl] glycine A mixture of 5-benzoyl-1,3-dimethyl- 2,4,6(lH,3H,5H)-pyrimidinetrione (519 mg, 2.00 mmol), glycine (100 mg, 1.33 mmol) and N,N-diisopropylethyl-amine (172 mg, 1.33 inmol) in abs. EtCH (10 ml) was stirred under ref lux overnight. The solvent was evaporated, the residue was taken up in CH 2 Cl 2 (100 ml) washed with 1 M KHS0 4 solution (10 ml) dried over MgSO 4 and evaporated. The residue was suspended with ether to give dimethyl-2, 4,6 (1H, 3H, 5H) -trioxopyrimidin-5-ylidene) phenylmethyl] glycine 15 (360 mg, 86%).

Rf 0.38 (CH 2 Cl 2 /EtOAc/MeOH 10:7:1) FAB MS C 15

H

15

N

3 0 5 (317 .29) m/z 318 (60) 272 130 (100).

WO 99/15510 WO 9915510PCT/AU98/00808 35 'H NNR (DMSO-d 6 d 12.30 1H, NH) 7.43 (in, 3H, 3 Ar-H) 7. 14 (in, 2H, 2 Ar-H) 3.76 2H, CH 2 3 .20, 2.93 (2s, 6H,1 2 NCH 3 Example 16 N-[l-(l,3-dimethyl-2,4,6(1H,3H,5H)phenylethyl] glycine 16 A mixture of 5-phenylacetyl-l,3-dimethyl- 2,4,6(lH,3H,5H)-pyrimidinetrione (548 mng, 2.00 inmol), glycine (100 mng, 1.33 mmol) and N,N-diisopropylethyl-amine (172 mg, 1.33 inmol) in abs. EtCH (10 ml) was stirred under ref lux overnight. The solvent was evaporated, the residue was taken up in CH 2 Cl 2 (100 ml) washed with 1 M KHS0 4 solution (10 ml) dried over MgSO 4 and evaporated. The residue was suspended with ether to give dimethyl-2,4,6 (lH, 3H, 5H) ylidene)phenylethyll glycine 16 (360 mg, 81%).

Rf 0 .4 0 (CH 2 C1 2 EtOAc /MeOH 10 1) FAB MS C1 6 H1 7

N

3 0 5 (331.32) m/z 354 [M+Na] 332 [M+H1+ (80) 286 (20) 130 (100) H NMR (CDCl 3 d 13.05 1H, NH) 7. 32 7.16 (in, Ar-H) 4. 69 2H, CH 2 Ar) 4. 14 2H, CH 2 3 .37, 3.29 (2s, 6H, 2 NCH 3 Example 17 Cleavage of 5-acyl-l,3-dimethylbarbituric acid protected primary amines affording 3-dimethylbarbituric acid protected hydroxylamines 34 N-[l-(l,3-Dimethyl-2,4,6(1H,3H, hydroxylamine 17 and Benzyl 2-deoxy-2-arnino-cL-D-glucopyranoside 34 Benzyl 2-deoxy-2-[l-(1,3-dimethyl-2,4,6(lH,3H,5H)-trioxophenylmethylamino] -x-D-glucopyranoside 22 (100 mg, 0.19 mmol) in NH 2 OH/MeOH 10 ml) was stirred at room temperature for 30 min. The solution was evaporated, the residue was suspended with ether (20 ml) WO 99/15510 WO 9915510PCT/AU98/00808 36 and filtered to give benzyl 2-deoxy-2-amino-aX-D-glucopyranoside 34 (45 mg, Rf 0.il (CH 2 Cl 2 /EtOAc/MeOH 10:7:3); FAB MS C1 3 H1 9 N0 5 (269.28) m/z M% 292 [M+Na1+ 270 (100), 253 178 (18).

1H NMR (DMSO-d 6 d 7. 35 7. 25 (in, 5H, 5 Ar-H) 4. 91, 4. 56 (2s, 2H, 2 NH) 4. 73 1H, H-1, J1, 2 3. 44 Hz) 4. 66, 4.40 (2d, 2H, CH 2 Ar) 3. 61 3 .05 (5 sugar-H) 2. 40 (dd, 1H, H-2).

The filtrate was evaporated and purified by chromatography using CH 2 Cl 2 /EtOAc/MeOH 10:7:1 to afford Nylidene)phenylmethyl] hydroxylamine 17 (40 mng, 73%).

Rf 0.76 (CH 2 Cl 2 /EtOAc/MeOH 10:7:3); FAB MS C1 3 H1 4

N

4 0 3 (275.25) m/z 298 IM+Na]+ (13), 276 (100), 243 'H NMR (ODC1 3 d 13. 95 1H, NH) 7. 32 7. 16 (in, Ar-H), 3.39, 3.14 (2s, 6H, 2 NCH 3 Example 18 N-[1-(1,3-dimethyl-2,4,6(lH,3H,5H)trioxopyrimidin- 5-yl idene) phenylmethyl] acetic hydrazide 18 A mixture of 5-benzoyl-l,3-dimethyl- 2,4,6(1H,3H,5H)-pyriinidinetrione 3 (260 mg, 1.00 mmiol) and acetic hydrazide (222 mg, 3.00 mmiol) in abs. ELON (10 ml) was stirred under ref lux overnight. The solvent was evaporated, the residue was taken up in CH 2 C1 2 (100 Ml), washed with 1 M KHS0 4 solution (10 ml) dried over MgSO 4 and evaporated. The residue was crystallized from MeOH to give N-[l-(1,3-dimethyl-2,4,6(1H,3H,5H)-trioxopyrimidin-5ylidene)phenylnethyll acetic hydrazide 18 (250 mg, 79%).

Rf 0.42 (MeCN/CHC1 3 2:1); WO 99/15510 WO 9915510PCT/AU98/00808 37 FAB MS C1 5 H1 6

N

4 0 4 (316.31) m/z M% 339 [M+Nal (28), 317 (100).

H NMYR (CDC1 3 d 13. 84 1H, NH) 7.61 1H, NH) 7.49, 7.20 (2m, 5H, 5 Ar-H) 3.38, 3 .13 (2s, 6H, 2 NCH 3 1.77 3H, NAG).

Example 19 Cleavage of 5-acyl-l,3-dimethylbarbituric acid protected primary amines affording acyl-1, 3-dimethylbarbituric acid protected hydrazines (1,3-Dimethyl-2, 4, 6 (1H, 3H, 5H) ylidene)phenylmethylj hydrazine 19 Benzyl 2-deoxy-2-[li- 3-dimethyl- 2,4,6 (1H, 3H, SH) -trioxopyimidin-5-ylidene) phenylmethylamino]-(X-D-glucopyranoside 22 (100 mg, 0.19 rnmol) in

N

2

H

4 /MeOH 10 ml) was stirred at room temperature for min. The solution was evaporated, the residue was suspended with ether (20 ml) and filtered to give benzyl 2-deoxy-2-amino-cu-D-glucopyranoside 34 (45 mg, Rf 0. 11 (CH 2 Cl 2 /EtOAc/MeOH 10: 7:3); The filtrate was evaporated, purified by chromatography using CH 2 C1 2 /EtOAc/MeOH 10:7:3 as the mobile phase to give N-[l-(1,3-dimethyl-2,4,6(lH,3H,5H)hydrazine 19 mg, 74%).

Rf 0.66 (CH 2 C1 2 /EtOAc/MeOH 10:7:3); FAB MS C1 3 H1 4

N

4 0 3 (274.25) m/z M% 297 (15) 275 (100), 243 'H NMR (CDCl 3 d 13.75 lH, NH) 7.32 7. 16 (in, Ar-H), 3.38, 3.13 (2s, 6H1, 2 NCH 3 WO 99/15510 WO 9915510PCT/AU98/00808 38 Example 20 Cleavage of 5-acyl-l,3-dimethylbarbituric acid protected primary amines with ammonia affording amino-substituted 5-acyl-l, 3dimethylbarbituric acid 5-Benzoimino-2, 3-dime thyl-2,4, 6 3H, 5H) -pyrimidinetri one Benzyl 2-deoxy-2-[1- (1,3-dimethyl- 2,4,6 (lH,3H, 5H) amino]-aX-D-glucopyranoside 22 (100 mg, 0.19 mmol) in 10 ml NH 3 /MeOH was stirred at room temperature for 30 min.

The solution was evaporated, the residue was suspended with ether (20 ml) and filtered to give benzyl 2-deoxy-2-amino- Ox-D-glucopyranoside 34 (48 mg, 92%).

Rf 0. 11 (CH 2 Cl 2 /EtOAc /MeOH 10: 7:3) The filtrate was evaporated to afford imino-l,3-dimethyl-2,4,6 (lH,3H, 5H) -pyrimidinetrione (47 mg, 93%).

Rf 0.86 (CH 2 Cl 2 /EtOAc/MeOH 10:7:3); FAB MS C 13

H

13

N

3 0 3 (259.25) m/z M% 282 260 (100), 243 1NMR (CDC1 3 d 12.48 1H, NH), 7.32 7.16 (in, 5 Ar-H) 3 .38, 3 .30 (2s, 6H, 2 NCH 3 Example 21 Cleavage of 5-acyl-l, 3-dimethylbarbituric acid protected primary amnines with primary amines NV-[l- (1,3-Dirnethyl 6(lH,3H,5H) yVlidene)phenylmethyl I 1-bu tylamine 4 Benzyl 2-deoxy-2-I[l-(l,3-dimethyl-2,4,6- (lH, 3H, 5H) -trioxopYrimidin-5-ylidene)phenylmethylamino] -a- D-glucopyranoside 22 (100 mg, 0.19 mmol) in 10 ml n-BuNH 2 was stirred at room temperature for 30 min. The solution was evaporated, the residue was suspended with ether WO 99/15510 WO 9915510PCT/AU98/00808 39 ml) and filtered to give benzyl 2-deoxy-2-amino-X-Dglucopyranoside 34 (48 mg, 92%).

Rf 0.-11 (CH 2 Cl 2 /EtOAc/MeOH 10:7:3); The filtrate was evaporated to afford dimethyl-2,4,6 (1H, 3H, 5H) -trioxopyrimidin-5-ylidene) phenylmethyl] 1-butylamine 4 (50 mg, 94%).

Rf 0. 89 (CH 2 Cl 2 /EtOAc /MeOH 10: 7:3); Example 22 Benzyl 2-deoxy-2-[l-(1,3-dimethyl- 2,4,6 (lH, 3H, 5H) ylidene) ethylamino] -o-D-glucopyranoside 21 A mixture of 5-acetyl-l,3-dimethyl-2,4,6- 1 (220 mg, 1.11 mmol), benzyl 2-amino-2-deoxy-c.-D-glucopyranoside 34 (200 mg, 0.74 mmol) and N,N-diisopropylethylamine (96 mg, 0.74 inmol) in abs.

ELOH (10 ml) was stirring under ref lux overnight. The solvent was evaporated, the residue was taken up in CH 2 Cl 2 (100 ml) washed with 1 M KHS0 4 solution (10 ml) The resulting suspension was filtered and the precipitate was washed with ether to give benzyl 2-deoxy-2-[1-(l,3dimethyl-2, 4,6- (lH, 3H, 5H) -trioxopyrimidin-5-ylidene) ethylaminoll-x-D-glucopyranoside 21 (245 mg, 73%).

Rf 0.43 (CH 2 Cl 2 /EtOAc/MeOH 10:7:3) FAB MS C 2 1

H

2 7

N

3 0 8 (449.45) m/z M% 472 (12), 450 (100) 358 (25) 342 (66).

1 H NM. (DMSO-d,) d 12.68 1H, NH) 7.46 2H, 2 Ar-H), 7.31 (in, 3H, 3 Ar-H), 4.95 1H, H-1, J 1 2 =3.60 Hz), 3.19, 3 .15 (2s, 6H, 2 NCHA), 2. 65 3H, CH 3 WO 99/15510 WO 99/ 5510PCT/AU98/00808 Example 23 Benzyl 2-deoxy-2-[l- (1,3-dimethyl- 2,4, 6(lH,3H,5H)-trioxopyrimidin-5ylidene) phenylmethylaminol -a-Dglucopyranoside 22 A mixture of 5-benzoyl-1,3-dimethyl-2,4,6- 3 (290 mg, 1.11 mmol), benzyl 2-amino-2-deoxy-a--D-glucopyranoside 34 (200 mg, 0.74 rnmol) and N,N-diisopropylethylamine (96 mg, 0.74 mmol) in abs.

ELOH (10 ml) was stirred under ref lux overnight. The solvent was evaporated, the residue was taken up in CH 2 Cl 2 (100 ml) washed with 1 M KHS0 4 solution (10 ml) and evaporated. The residue was crystallized from MeCN to give benzyl 2-deoxy-2-[1-(1,3-dimethyl-2,4,6(1H,3H,5H)- -phenylmethylamino] -a-Dglucopyranoside 22 (270 mg, 71%).

Rf 0.35 (CH 2 01 2 /EtOAc/MeOH 10:7:1); FAB MS C 2 6

H

2 9

N

3 0 8 (511.51) m/z M% 534 [M+Na] 4 (18) 512 [M+Hl 4 (100) 420 (18) 404 (36) 338 (75) 1 H NMR (DMSO-d 6 d 12.47 1H, NH) 7.41 7.17 (in, Ar-H), 4.66 1H, H-1, J 1 2 =3.55 Hz), 4.68, 4.48 (2d, 2H, CH 2 Ar) 2. 99, 2. 94 (2s, 6H, 2 NCH 3 Example 24 Benzyl 2-deoxy-2-Ijl-(l,3-dimethyl- 2 4 6 (lH,3H,5H)-trioxopyrimidin-5-ylidene) (9fluorenylmethylamino)] -OC-D-glucopyranoside 23 A mixture of 5- (9-f luorenylcarbonyl) -1,3dimethyl-2,4,6(1H,3H,5H)-pyrimidinetrione 5 (388 mg, 1.11 mmol) and benzyl 2 -amino-2-deoxy-cx-D-glucopyrano-side 34 (200 mg, 0.74 mmol) in abs. EtOH (10 ml) was stirred under ref lux overnight. The solvent was evaporated, the residue was taken up in CH 2 C1 2 (100 ml) washed with 1 M KHS0 4 solution (10 ml) and evaporated. The residue was purified by chromatography using CHCl 3 /MeCN/AcOH 10:10:0.1 to give benzyl 2 -deoxy-2-II1-(1,3-dimethyl-2,4,6(1H,3H,5H)- (9-f luorenylmethylamino) I-aX-Dglucopyranoside 23 (140 mg, 31%).

WO 99/15510 WO 9915510PCT/AU98/00808 -41- Rf 0. 37 (CHCl 3 /NeCN/AcOH 10: 10: FAB MS C 3 3

H

3 3

N

3 0 8 (599. 61) m/z M% 622 [M+Nal (48), 600 (100) 492 (88) 474 (26) 346 1H NMR (CDCl 3 d 12.72 1H, NH) 7.85 -6.77 (in, 14H, 13 Ar-H, CH) 4. 57, 4.22 (2d, 2H, CH 2 Ar), 3.47, 3.40 (2s, 6H, 2 NCH 3 Example 25 Benzyl 2-deoxy-2- [1-(1,3-dimethyl- 2,4,6(lH,3H,5H)-trioxopyrimidin-5ylidene) phenylethylamino] -cL-D-glucopyranoside 24 A mixture of 5-phenylacetyl-l,3-dimethyl- 2,4,6(1H,3H,5H)-pyrimidinetrione 8 (305 mg, 1.11 mmol), benzyl 2-amino-2-deoxy-aX-D-glucopyranoside 34 (200 mg, 0.74 mmol) and N,N-diisopropylethylamine (96 mg, 0.74 mmol) in abs. ELOH (10 ml) was stirred under ref lux overnight.

The solvent was evaporated, the residue was taken up in

CH

2 Cl 2 (100 ml) washed with 1 M KHS0 4 solution (10 ml) and evaporated. The residue was purified by chromatography using CHCl 3 /EtOAc/MeOH 10:7:1 as the mobile phase to give benzyl 2-deoxy-2-[1-(l,3-dimethyl-2,4,6(lH,3H,5H)- -phenylethylamino] -ci-Dglucopyranoside 24 (280 mg, 72%).

Rf 0.47 (CHCl 3 /EtOAc/MeOH 10:7:1); FAB MS C 2 7

H

3 1

N

3 0 8 (525. 54) m/z 548 [M+Na] (22), 526 (100) 417 274 (47).

1 HNIAR (DMSO-d 6 d 12.88 1H, NH) 7.41 7. 01 (in, 10 Ar-H) 4. 65, 4.39 (2d, 2H, CH 2 Ar) 4.38 1H, H-1, J1, 2 =3.03 Hz) 3.23, 3.09 (2s, 6H, 2 NCH 3 WO 99/15510 WO 9915510PCT/AU98/00808 42 Example 26 Berizyl 2-deoxy-2-[1- (1,3-dimethyl- 2,4,6 (1H, 3H, 5H) ylidene) diphenylethylamino] -a-Dglucopyranoside A mixture of 5-diphenylacetyl-l,3-dimethyl-- 2,4,6(lH,3H,5H)-pyrimidinetrione 9 (390 mg, 1.11 mmol), benzyl 2-amino-2-deoxy-ax-D-glucopyranoside 34 (200 mg, 0.74 mmol) and N,N-diisopropylethylanine (96 mg, 0.74 rumol) in abs. EtOH (10 ml) was stirred under ref lux overnight.

The solvent was evaporated, the residue was taken up in

CH

2 C1 2 (100 ml) washed with 1 M KHS0 4 solution (10 ml) and evaporated. The residue was purified by chromatography using l,2-dichloroethane-/MeOH/AcOH 10:1:0.1 as the mobile phase to give benzyl 2-deoxy-2-Ijl-(l,3-dimethyl- 2,4,6(lH,3H,5H)-trioxo-pyrimidin-5ylidene) diphenylethylamino] -c-D-glucopyranoside 25 (300 mg, 68%).

Rf 0.37 (1,2-dichloroethane/MeOH/AcOH 10:1:0.1); FAB MS C 33

H

3 5

N

3 0 8 (601.63) m/z M% 624 602 (100) 494 (47) 348 (42) 338 (39) 1 H NMR (CDCl 3 d 13.44 1H, NH) 8.15 1H, CHAr 2 7.52 6.94 (in, 15H, 15 Ar-H) 4.55, 4.21 (2d, 2H, CH 2 Ar), 3 .39, 3 .29 (2s, 6H, 2 NCH 3 Example 27 Benzyl 2-deoxy-2- [1-(1,3-diinethyl- 2,4,6 (lH, 3H, 5H) -trioxopyrimidin-5-ylidene) 2-dime thylpentylamino)]I-aX-Dglucopyranoside 28 A mixture of 5-pivaloyl-l,3-dimethyl- 2,4,6(lH,3H,5H)-pyrimidinetrione 11 (267 mng, 1.11 mmol), benzyl 2-amino-2-deoxy-cX-D-glucopyranoside 34 (200 mg, 0.74 mmol) and N,N-diisopropylethylamine (96 mg, 0.74 minol) in abs. EtOH (10 ml) was stirring under ref lux overnight.

The solvent was evaporated, the residue was taken up in

CH

2 Cl 2 (100 ml) washed with 1 M KHS0 4 solution (10 ml) and evaporated. The residue was purified by chromatography WO 99/15510 WO 9915510PCT/AU98/00808 43 using CH 2 Cl 2 /EtOAc/MeOH 10:7:3 as the mobile phase to give berazyl 2-deoxy-2-[l-(l,3-dimethyl-2,4,6(lH,3H,5H)- 2-dimethylpentylamino) -a-Dglucopyranoside 28 (240 mg, 66%).

Rf 0.47 (CH 2 Cl 2 /EtOAc/MeOH 10:7:3); FAB MS C 2 4

H

3 3

N

3 0 8 (491. 52) m/z 514 EM+Na] (28) 492 (100) 270 (25) 240 (54).

1H INMR (CDCl 3 d 12.76 1H, NH) 7.29 (in, 5H, 5 Ar-H) 4. 64, 4. 40 (2d, 2H, CH 2 Ar) 3 .24, 3 .21 (2s, 6H, 2 NCHA) 1.37 9H, 3 CH 3 Example 28 Benzyl 2-deoxy-2- 3-dimethyl- 2,4,6 (1H, 3H, 5H) -trioxopyrimidin-5-ylidene) (1-adamantylmethylanino) ]-0X-D-glucopyranoside 29 A mixture of 5-adamantanecarbonyl-1, 3-dimethyl- 2,4,6(1H,3H,5H)-pyrimidinetrione 12 (709 mg, 2.23 ramol), benzyl 2-amino-2-deoxy-aX-D-glucopyranoside 34 (200 mng, 0.74 inmol) and N,N-diisopropylethylamine (288 mng, 2.23 minol) in abs. EtOH (10 ml) was stirred under ref lux overnight. The solvent was evaporated, the residue was taken up in CH 2 Cl 2 (100 ml) washed with 1 M KHS0 4 solution ml) and evaporated. The residue was suspended with ether to give benzyl 2-deoxy--2-[l-(l,3-dimethyl- 2,4,6 (lH, 3H, 5H) -trioxopyrimidin-5-ylidene) (-adamantylmethylamino)]-cx-D-glucopyranoside 29 (260 mng, 62%).

Rf 0.45 (CH 2 Cl 2 /EtOAc/MeOH 10:7:3) FAB MS C 3 0

H

3 9

N

3 0 8 (569. 63) m/ z M% 592 [M+Na] 570 [M+H1+ (100).

H NMR (CDCl 3 d 12.74 1H, NH) 7.33 5H, 5 Ar-H) 4.65, 4.43 (2d, 2H, CH 2 Ar) 3 .27, 3 .22 (2s, 6H, 2 NCHA) 2.13, 2.04 (2s, 12H, 6 CH 2 1.72 (mn, 3H, 3 CH) WO 99/15510 PCT/AU98/00808 -44- Example 29 Reaction of primary amines with trichloroacetimino-1,3-direthyl- 2,4,6(lH,3H,5H)-pyrimidinetrione Benzyl 2-deoxy-2-[1-(1,3-dimethyl-2,4, 6(1H,3H,5H) trioxopyrimidin-5-ylidene) ainomethyl amino] -a-Dglucopyranoside 27 A mixture of 5-Trichloroacetimino-1,3-dimethyl- 2,4,6(lH,3H,5H)-pyrimidinetrione 13 (333 mg, 1.11 nmol), benzyl 2-amino-2-deoxy-CX-D-glucopyranoside 34 (200 mg, 0.74 nmol) and N,N-diisopropylethylaine (96 mg, 0.74 nmol) in abs. EtOH (10 ml) was stirred under reflux overnight.

The solvent was evaporated, the residue was taken up in

CH

2 C1 2 (100 ml), washed with 1 M KHS0 4 solution (10 ml) and evaporated. The residue was purified by chromatography using CH 2 Cl 2 /EtOAc/MeOI 10:7:3 as the mobile phase to give benzyl 2-deoxy-2-[l-(l,3-dimethyl-2,4,6(lH,3H,5H)glucopyranoside 27 (250 mg, Rf 0.41 (CH 2 Cl 2 /EtOAc/MeOH 10:7:3); FAB MS C 2 0

H

2 6

N

4 0 8 (450.44) m/z 473 [M+Nal 4 (21), 451 (100), 358 342 265 (269).

1H NMR (DMSO-d 6 d 10.86 1H, NH), 10.06 1H, NH), 7.74 1H, NH), 7.42 2H, 2 Ar-H), 7.29 3H, 3 Ar-H), 4.87 1H, H-l, J1, 2 3.22 Hz), 4.69, 4.48 (2d, 2H, CH 2 Ar).

Example 30 Preparation of "Linker-Carbohydrate Conjugate' Benzyl 2-deoxy-2-[1-(1,3-dimethyl-2,4, 6(1H,3H,5H) (4-carboxybutylamino)] -u-Dglucopyranoside 26 A mixture of 5-(4-carboxybutyryl)-1,3-dimethyl- 2,4,6(lH,3H,5H)-pyrimidinetrione 10 (301 mg, 1.11 nmol), benzyl 2-amino-2-deoxy-U-D-glucopyranoside (200 mg, 0.74 mmol) and N,N-diisopropylethylamine (240 mg, 1.85 nmol) in abs. ELOH (10 ml) was stirred under reflux WO 99/15510 PCT/AU98/00808 overnight. The solvent was evaporated, the residue was taken up in CH 2 C1 2 (100 ml) and washed with 1 M KHS04 solution (10 ml). The resulting suspension was filtered, the precipitate was washed with ether giving benzyl 2-deoxy-2-[l-(l,3-dimethyl-2,4,6(lH,3H, 5-ylidene)(4-carboxybutylamino)]-cx-D-glucopyranoside 26 (280 mg, 73%).

Rf 0.28 (CH 2 C1 2 /EtOAc/MeOH 10:7:5); FAB MS C 2 4H 3 1

N

3 01 0 (521.51) m/z 544 522 [M+H1 (100), 430 414 1H NMR (DMSO-d 6 d 12.70 1H, NH), 7.45 7.18 Ar-H), 4.97 1H, H-1, J1, 2 =3.47 Hz), 4.97, 4.4.72 (2d, 2H, CH 2 Ar), 3.17, 3.14 (2s, 6H, 2 NCH 3 3.00 2H, CH 2 2.34 4H, 2 CH 2 Example 31 Chiral 5-acyl-1,3-dimethylbarbituric acid derivatives for primary amine protection N,N'-Bis-(benzyl 2-deoxy-c-D-glucopyranosid-2-yl)-[5-(2aminoacetimino)-1,3-dimethyl-2,4, 6(1H,3H,5H) pyrimidinetrionej 30 and 5-[N-(benzyl 2-deoxy-ci-Dglucopyranosid-2-yl)ainoacetyl]-1,3-dimethyl- 2,4, 6 (1H, 3H, 5H) -pyriridinetrione 31 A mixture of 5-chloroacetyl-,3-diethyl- 2,4,6(lH,3H,5H)-pyrimidinetrione 6 (260 mg, 1.11 mmol), benzyl 2 -amino-2-deoxy-a-D-glucopyranoside 34 (200 mg, 0.74 mmol) and N,N-diisopropylethylamine (96 mg, 0.74 nmol) in abs. EtOH (10 ml) was stirred under reflux overnight.

The solvent was evaporated, the residue was taken up in

CH

2 Cl 2 (100 ml), washed with 1 M KHS0 4 solution (10 ml) and evaporated. The residue was purified by chromatography using CH 2 Cl 2 /EtOAc/MeOH 10:7:3 as the mobile phase to give N,N'-bis-(benzyl 2-deoxy-a-D-glucopyranosid-2-yl)-[5-(2aminoacetimino)-1,3-dimethyl-2,4,6(1H,3H,5H)pyrimidinetrione] 30 (110 mg, 21%).

Rf 0.42 (CH 2 Cl 2 /EtOAc/MeOH 10:7:3); WO 99/15510 WO 9915510PCT/AU98/00808 -46 FAB MS C 3 4

H

4 4

N

4 01 3 (716 .72) m/z M% 739 [M+Naf (22), 717 (100).

1H NIMR (DMSO-d 6 d 12.58 1H, NH), 7.43 7.25 (in, Ar- H) 4. 65 4. 24 (4d, 4H, 2 CH 2 Ar) 3 .18, 3. 08 (2s, 6H, 2 NCH 3 arnd 5-[IN-(benzyl 2-deoxy-aX-D-glucopyranosid-2yl)aminoacetyl]-1,3-dimethyl-2,4,6(lH,3H,5H)pyrimidinetrione 30 (80 mg, 23%).

Rf 0.33 (CH 2 Cl 2 /EtOAc/MeOH 10:7:3); FAB MS C 2 1

H

2 7

N

3 0 9 (465. 45) m/z M% 488 [M+Na] (27), 466 (100).

1H NIMR (DMSO) d 17.22 1H, OH), 7.41 7.27 (mn, Ar-H), 4.68, 4.46 (2d, 2H, CH 2 Ar), 3.19, 3.14 (2s, 6H, 2 NCH 3 Example 32 Preparation of resin-linker-carbohydrate conjugate Benzyl 2-deoxy-2-[2-(1,3-dimethyl-2,4, 6(lH,3H, (4 -carboxybutyl amino) 1-3,4, 6-tnr- O-acetyl-ct-D-glucopyranoside M.BHA resin conj ugate 32 Benzyl 2-deoxy-2- 3-dimethyl- 2,4,6(1H,3H,5H) -trioxopyrimidin-5-ylidene) (4carboxybutylamino) I-aX-D-glucopyranoside 26 (300 mng, 1.11 inmol) was dissolved in pyridine (10 ml), cooled to 0 0

C

and acetic anhydride (7 ml) added. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated and the resulting residue was taken up in CH 2 Cl 2 ml), washed with 1 M KHS0 4 solution, dried over MgSO 4 and evaporated. The residue was taken up in DMF (10 ml) and was used as a reagent during the resin work. MBHA resin (Subst. ratio: 0.42 rnmol/g) (200 ing) bearing a total amine functionality of 0.084 inmol was swelled in DMF for min. The resin was then washed with fresh DMF and berizyl 2-deoxy-2-[l-(l,3-dimethyl-2,4,6(lH,3H,5H)trioxopyriinidin-5-ylidene) (4-carboxybutylanino) 1-3,4, 6-tni- O-acetyl-cX-D-glucopyranoside DMF solution (5 ml, 6.6 equiv.) and N,N'-diisopropylcarbodiinide (88 ml, WO 99/15510 PCT/AU98/00808 -47- 6.6 equiv.) were added and the resin gently agitated for min. The TNBS test was faintly positive so using the above conditions, a double coupling was performed, this time producing a negative TNBS test result. The resin was washed with DMF, methanol and finally ether. The resin was then allowed to dry in vacuum over KOH overnight.

Example 33 Carbohydrate deprotection and cleavage of the "fully protected sugar linker resin conjugate" providing an "amino substituted resin linker conjugate" The resin from Example 32 was gently agitated with sodium methoxide (100 mg, 1.85 mmol) in abs. MeOH ml) at room temperature for 1 h. The resin was washed with abs. MeOH (5x10 ml), DMF(5xlO ml), ether (5x10 ml) and dried under high vacuum for 1 h, giving the resin bonded unprotected benzyl 2-amino-2-deoxy-a-D-glucopyranoside. A sample of resin (5 mg) was cleavaged by saturated NH 3 /MeOH (0.2 ml) at room temperature for 10 min. The resin was filtered off, the filtrate was evaporated giving benzyl 2-amino-2-deoxy-a-D-glucopyranoside 34 in quantitative yield. During the cleavage conditions the resin was transformed into its amino-substituted form Example 34 Preparation of "resin-linker-carbohydrate conjugate" using "amino-substituted resinlinker conjugate" "Amino-substituted resin-linker conjugate" (100 mg, 0.042 mmol amine functionality), benzyl 2-amino-2deoxy-a-D-glucopyranoside 34 (34 mg, 0.13 mmol) and diisopropylethylamine (16 mg, 0.126 mmol) in abs. EtOH gently stirred under reflux overnight. The reaction mixture was filtered, the resin was washed with MeOH, DMF,

CH

2 C1 2 ether and dried to give the "resin-linkercarbohydrate conjugate" 37.

WO 99/15510 PCT/AU98/00808 -48- Example 35 Preparation of a "hydroxy-substituted resinlinker conjugate" 36 MBHA resin (Subst. ratio: 0.42 mmol/g) (200 mg) bearing a total amine functionality of 0.084 mmol was swelled in DMF for 20 min. The resin was then washed with fresh DMF and 5-(4-carboxybutyryl)-1,3-dimethyl- 2,4,6(1H,3H,5H)-pyrimidinetrione 10 (68 mg, 0.25 mmol) and N,N'-diisopropylcarbodiimide (40 ml, 3.0 equiv.) were added in DMF (5 ml) and the resin gently agitated for 30 min.

The TNBS test was faintly positive so using the above conditions, a double coupling was performed, this time producing a negative TNBS test result. The resin was washed with DMF, methanol and finally ether. The resin was then allowed to dry in vacuum over KOH overnight to give 36.

Example 36 Preparation of a "hydroxy-substituted resinlinker conjugate" using "amino-substituted resin-linker conjugate" 36 "Amino-substituted resin-linker conjugate" mg, 0.021 mmol amine functionality) was stirred at room temperature in 1 M NaOH solution (2.0 ml) for 10 min. The mixture was filtered, washed with H 2 0, methanol and finally ether. The resin was then allowed to dry in vacuum over KOH overnight to give 36.

Example 37 Preparation of "2-acetyl-l,3-indanedione" 38 A mixture of 4-dimethylaminopyridine (664 mg, 5.44 mmol), triethylamine (7.6 ml 54.56 mmol), acetic anhydride (6.2 ml, 65.48 mmol in dry 1,2-dichloroethane ml) was stirred at -20 0 C and a solution of 1,3-indanedione (7.96 g, 54.56 mmol) in 1,2-dichloroethane was added dropwise in 1.5 h. The reaction mixture was stirred for 30 min, then washed with 10% hydrochloric acid (80 ml) and twice with water (80 ml). The organic phase was dried over MgS0 4 and evaporated. The residue was WO 99/15510 PCT/AU98/00808 -49crystallized from methyl-tert-butylether (50 ml) to give 2-acetyl-l,3-indanedione 38(6.5 g Rf 0.27 (hexaneethylacetate-acetic acid 20-5-0.5) MS C 11 H03 m/z 189 (100), 166 104 Example 38 Methyl2-deoxy-2-[1-(1,3-dimethyl- 2,4,6(1H,3H,5H)-trioxopyrimidin-5ylidene)ethylamino]-l-thio-b-Dglucopyranoside 39 Methyl 2-deoxy-2-amino-l-thio-P-D-glucopyranoside (5.00 g, 23.9 mmol) was dissolved in dry ethanol (70 ml) and 1,3-dimethylbarbituric acid (9.47 g, 47.8 mmol) added to form a suspension. Triethylamine (5.40 g, 53.3 mmol) was then added and the resultant clear solution heated at reflux for 14h. The solvent was evaporated, the residue dissolved in dichloromethane (200 ml) and 5% hydrochloric acid solution (200 ml) added. The resultant precipitate was collected and recrystallized from ethyl acetate to yield Methyl 2-deoxy-2-[1-(1,3-dimethyl-2,4,6(1H,3H,5H)trioxopyrimidin-5-ylidene)ethylamino]-1-thio-P-Dglucopyranoside 39, as a colourless solid (7.82 g, 84.1 Rf 0.57 (CH 3

CN/H

2 0 9:1); ESI-MS MS m/z 390.0 H NMR (CDC1 3 d 4.650 3H, J 1 ,2=9.9 Hz, H1), 3.894 (dd, 1H, 3.716 (dd, 1H, 3.547 (dd, 1H, 3.426 2H, 3.306 1H, 3.266 6H, 2 x N-CH 3 2.730 3H, vinylic-CH 3 2.211 3H, S-CH 3 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 WO 99/15510 PCT/AU98/00808 from the scope of the inventive concept disclosed in this specification.

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

t% WO 99/15510 WO 99/ 5510PCT/AU98/00808 51

REFERENCES

Bergman, M. and Zervas, L.

Ber. Dtsc. Chem. Ges., 1932 65 1192 Branchaud, B.P.

J. Org. Chem., 1983 48 3538.

Bycroft, Chan, Chhabra, Teesdale-Spittle, S.R. and Hardy, P.H.

J. Chem. Soc. Chem Commun., 1993 777.

Bycroft, Chan, Chhabra, S.R. and Hone, N.D.

J. Chem. Soc. Chemn. Commun., 1993 778.

Carpino, Tsao, Ringsdorf, Fell, E. and Gettrich, J.G.

J. Chem. Soc. Chem. Common., 1978, 358.

Chan, Bycroft, Evans, D.J. and White, P.D.

J. Chem. Soc. Chem. Comun., 1995 2209.

Colvin, McGarry, D. and Nugent, M.J.

Tetrahedron Lett., 1988 44 4157.

Fischer, E. and Livschitz, W.

Ber. Dtsch. Chem. Ges., 1915 48 360.

Goerdeler, J. and Hoist, A.

Angew. Chem., 1959 71 775.

Goldstein, Overman, L.E. and Rabinowitz, M.H.

J. Org. Chem., 1992 57 1179.

Gribble, Saulnier, Obaza-Nutaitis, J.A. and Ketcha, D.M.

J. Org. Chem., 1992 57 1581.

WO 99/15510 WO 9915510PCT/AU98/00808 -52 Halpern, B. and James, L.B.

Aust. J. Chem., 1964 17 1282.

Hoppe. D. and Beckmann, L.

Liebigs Ann. Chem., 1979 2066.

Kellam, B.

Ph.D. Dissertation, 1996.

Kessler, W. and Iselin, B.

Helv. Chim. Acta., 1966 49 1330.

Koskinen, A.M. and Rapoport, H.

J. Org. Chem., 1989 54 1859.

Kunz, H. and Unverzagt, C.

Angew. Chem. Int. Ed. Eng., 1984 23 436 McKay, F.C. and Albertson, N.F.

J. Am Chem. Soc., 1957 79 4686 Mosher, W.A. and Meier, W.E.

J. Org. Chem., 1970 35 2924.

Nicolaou, Bockovich, N.J, Carcanague, Humnmel, C.W. and Iven, L.F.

J. Am. Chem. Soc., 1992 114 8701 Overman, Okazaki, M.E. and Mishra, P.

Tetrahedron Lett., 1986 27 4391.

Polt, Szabo, Treiberg, Li, Hruby, V.J.

J. Am. Chem. Soc., 1992 114 10249.

Sieber, P. and Riniker, B.

Tetrahedron Lett., 1991 32 739.

WO 99/15510 PCT/AU98/00808 53 Weinreb, Demko, Lessen, T.A. and Demers. J.P.

Tetrahedron Lett., 1986 27 2099 Weygand, F. and Czendes, E.

Angew Chem., 1952 64 136 Windholz, T.B. and Johnston, D.B.R.

Tetrahedron Lett., 1967 2555.

Claims (28)

1. A compound of general formula I R 0 N Ri 04_ N R2 /R R 0 I in which each R is independently H; substituted or unsubstituted alkyl, aryl, alkenyl or alkynyl; or acyl; R 1 is hydrogen; an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloheteroaryl, cycloalkyl, heterocycloalkyl, alkanal, or thioalkanal group, each of which may be unsubstituted or substituted S* with substituents selected from amino, azido, halogen, hydroxyl, guanidino, carboxy, carboxylic acid ester, thio, carboxamide, alkylamino, dialkylamino, trialkylammonium, and alkoxy; and when R' is hydrogen, then R 2 is a protected, unprotected or substituted sugar amino-; a glycosylamino-, or a glycosylamino group of an S* oligosaccharide; a mono- or oligosaccharide coupled through a substituted or unsubstituted alkylamino-, 25 arylamino-, cycloalkylamino, heteroalkylamino, heteroarylamino or heterocycloalkylamino group; an amino sugar; an amino acid or a peptide linked via a nitrogen atom; and when R 1 is an alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloheteroaryl, cycloalkyl, heterocycloalkyl, alkanal, or thioalkanal group, each of which may be substituted or unsubstituted then R 2 is a protected, unprotected or substituted sugar amino-; a glycosylamino-, or a glycosylamino group of an oligosaccharide; a mono- or Soligosaccharide coupled through a substituted or g ^unsubstituted alkylamino-, arylamino-, H:WiMarnnclKU(S|iao\Q3303-98.1 SPICdocd 29/10)02 29/10 2002 15:05 FAX 61 3 92438333 GRIFFITH HACK @013 55 cycloalkylamino, heteroalkylamino, heteroarylamino or heterocycloalkylamino group; an amino sugar; an amino acid or a peptide linked via a nitrogen atom; an alkylamino, dialkylamino, arylamino, or diarylamino group, each of which may be substituted or unsubstituted, with the provisos that a) when R 1 is unsubstituted phenyl, then R 2 is not unsubstituted aminophenyl (aniline) and b) when R 1 is methyl, then R 2 is not alkylamino substituted with a compound of formula I in which R is hydrogen and R 1 is methyl.

2. A compound according to Claim 1, in which each R group has 1 to 6 carbon atoms.

3. A compound according to Claim 1 or Claim 2, in which each R group has 2 to 4 carbon atoms.

4. A compound according to any one of Claims 1 to 3, in which R 1 is selected from chloromethyl, trichloromethyl, and derivatives thereof.

A compound according to any one of Claims 1 to 4, which is chiral.

6. A compound according to any one of Claims 1 to 5, in which R 2 is an amino sugar; a glycosylamino-, or a glycosylamino group of an oligosaccharide; a mono- or oligosaccharide coupled through a substituted or unsubstituted alkylamino-, arylamino-, cycloalkylamino, heteroalkylamino, heteroarylamino or heterocycloalkylamino group; or an amino acid or a peptide linked via a nitrogen atom. Hamunplel\Keqectw133.398.1 SrECIldom 29/10 29/10 2002 15:05 FAX 61 3 92438333 GRIFFITH HACK @014 56

7. A compound according to any one of Claims 1 to 5, of general formula IVb: R N- R R °*o o oooo o o oooo oooo *oo *ooooo IVb in which R and R 1 are as defined in Claim 1; and R 2 b is a protected, unprotected or substituted sugar amino-; a glycosylamino- or a glycosylamino group of an oligosaccharide; or a mono- or oligosaccharide coupled through a substituted or unsubstituted alkylamino-, 10 arylamino-, cycloalkylamino, heteroalkylamino, heteroarylamino or heterocycloalkylamino group.

8. A compound according to any one of Claims 1 to 7, of general formula IVc: R 0 N R' N R 2 R O IVc in which R and R 1 are as defined in claim 1; and R c is an oligosacharide-O-CH 2 (CH 4 monosaccharide-O-CH- (C6H4)-NH-, oligosaccharide-CO2CH2- (C6H4)NH-, or monosaccharide-C02CH2-(C6H 4 )-NH group. HAimanUKelepSpe dkM3-9x.I .SPIECIdoc 29/1002 29/10 2002 15:05 FAX 61 3 92438333 GRIFFITH HACK [015 57

9. A support of general formula VI for solid-phase synthesis of oligosaccharides, peptides or organic compounds, comprising a resin and a linker covalently attached to the resin R o Rla-RESIN N A 0 VI in which R is as defined in Claim 1; and R is a substituted or unsubstituted alkyl, 10 cycloalkyl, heteroalkyl, heteroaryl, heterocycloalkyl or carboxylamido spacer group which is directly coupled to o the resin, or which may optionally be coupled to the resin via a suitable covalent linkage, which is stable to conditions of oligosaccharide synthesis and cleavage, and 15 R 2 is a protected, unprotected or substituted sugar amino-; a glycosylamino-, or a glycosylamino group of an oligosaccharide; a mono- or oligosaccharide coupled through a substituted or unsubstituted alkylamino-, arylamino-, cycloalkylamino, heteroalkylamino, S. 20 heteroarylamino or heterocycloalkylamino group; an amino sugar; an amino acid or a peptide linked via nitrogen atom; an alkylamino, dialkylamino, arylamino, or diarylamino group, each of which may be substituted or unsubstituted; OH, 0-M, O-alkyl, 0-acyl, O-aryl, each of which may be substituted or unsubstituted; and M is a metal ion, or an organic or inorganic cation.

II:sunnneU= CeeAprSpcc 33O3--A,1 SPEiJOc 291WO02 29/10 2002 15:05 FAX 61 3 92438333 GRIFFITH HACK @016 58 A support according to Claim 9, in which R 2 is as defined in Claim 1.

11. A support according to Claim 9 or Claim 10, in which the covalent linkage is provided by a -CONH-, NH-, -COO-, -COS-, -NHCONH-, -NHCSNH or -NHNH- grouping.

12. A support according to any one of Claims 9 to 11, in which the resin swells in water and/or in an organic solvent, and which comprises one of the following substituents: halogen, hydroxy, carboxyl, SH, NH2, formyl, S0 2 NH 2 or NHNH 2 15

13. A method of solid-phase synthesis of oligosaccharides, comprising the step of sequentially linking mono- or oligosaccharide groups to the support according to any one of Claims 9 to 12.

14. A method according to Claim 13, in which 2 a) the linker is synthesised directly on the resin in a stepwise manner prior to the coupling of the initial sugar group, or b) the linker-initial sugar conjugate is 25 synthesised in solution phase and subsequently coupled to the support, with subsequent sugars being sequentially attached.

A method according to Claim 13 or Claim 14, in which the support comprises a resin, a linker and a saccharide selected from the group consisting of monosaccharides, oligosaccharides, aminosaccharides and aminooligosaccharides.

16. A method according to any one of Claims 13 to 15, in which the second and subsequent sugar groups are coupled Sto the oligosaccharide chain-resin conjugate after the Hnlaun MColKApSpct93.3-.9A.1 SpAJ,doc ;9Z/IV02 29/10 2002 15:06 FAX 61 3 92438333 GRIFFITH HACK 1017 59 last sugar in the oligosaccharide chain is partially deprotected.

17. A method according to any one of Claims 13 to 16, in which the first sugar attached to the resin-linker unit is an unprotected, partially protected or fully protected glycoside, aminoglycoside, ether-linked sugar or amino-linked sugar.

18. A method according to Claim 17, in which the first sugar coupled to the resin is an aminosugar, an aminoglycoside or an amino-oligosaccharide, or a glycosyl amine of an oligosaccharide. 15

19. A method according to any one of Claims 13 to 18, in which the oligosaccharide is branched, and deprotection is achieved by using one or more protecting groups selected from the group consisting of acyl-type, trityl, methoxytrityl, methoxybenzyl, silyl and photolabile 20 protecting groups in addition to permanent ether-type protecting groups.

20. A reagent for solution phase synthesis of sugar- containing compounds, comprising the barbituric acid 25 derivative compound according to any one of Claims 1 to

21. A linker-saccharide complex, comprising a linker group and a protected saccharide compound according to any one of Claims 6 to 8.

22. A method of solution phase synthesis of oligosaccharides, comprising the step of sequentially linking mono- or oligosaccharide groups to the compound according to any one of Claims 6 to 8 or 21.

23. A method according to Claim 22, in which combinatorial synthesis of aminoglycosides is performed. H sunneLU~saq pcdfllw-' I SPECIAdC 29/1W 29/10 2002 15:06 FAX 61 3 92438333 GRIFFITH HACK 018 60

24. A kit useful in solid phase synthesis or combinatorial synthesis, comprising a) the support according to any one of Claims 9 to 12, or b) the compound according to any one of Claims 6 to 8, and optionally also comprising one or more further reagents and/or solvents suitable for solid phase or combinatorial synthesis.

25. A kit for solution phase synthesis or combinatorial synthesis of oligosaccharides, comprising the compound according to any one of Claims 6 to 8 or 20, and 15 optionally also comprising one or more further reagents and/or solvents suitable for solid phase or combinatorial synthesis.

26. A kit according to Claim 24 or Claim 25, in which the further reagents are protecting and/or deprotecting agents.

27. Use of a reagent for solution phase synthesis of sugar-containing or amino acid containing compounds, said 25 reagent comprising a barbituric acid compound of formula I in which R and R 1 are as defined in claim 1 and R 2 is selected from a protected, unprotected or substituted or unsubstituted sugar amino-; a glycosylamino-;or a glycosylamino group of an oligosaccharide; a mono- or oligosaccharide coupled through a substituted or unsubstituted alkylamino-, arylamino-, cycloalkylamino, heteroalkylamino, heteroarylamino or heterocycloalkylamino group; an amino sugar; an amino acid or a peptide linked via nitrogen atom, an alkylamino, dialkylamino, or diarylamino group, each of which may be substituted or unsubstituted; OH, O-M,0-alkyl, O-acyl, 0-aryl and M is a metal ion, or an organic or inorganic cation. Itcflcc~bcanspedt93o34fl.I Sp.Ct'doc flh/1J 29/10 2002 15:06 FAX 61 3 92438333 GRIFFITH HACK 019 61

28. Compounds of general formulae I or VI, processes for their preparation or their use in solid-phase or solution phase synthesis of oligosaccharides or combinatorial synthesis of aminoglycosides, substantially as hereinbefore described with reference to the examples Dated this 29th day of October 2002 ALCHEMIA PTY LTD By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and 15 Trade Mark Attorneys of Australia *9 *9* 9 *9. H:;Wuwn$JCvAa 5pvc~H3'3O-f9LA.I SPfC-Ldac 2tI(YOZ