AU3060600A - Inositol-containing hexasaccharides, their synthesis and their uses - Google Patents

Description

WO 00/39141 PCT/GB99/04400 Inositol-Containincr Hexasaccharides, Their Synthesis and Their Uses Field of the Invention 5 The present invention relates to novel inositol containing hexasaccharides, in particular to hexasaccharides capable of acting as inositol phosphoglycan (IPG) mimetics. It also relates to the synthesis of such hexasaccharides, to intermediate 10 compounds formed during their synthesis, to the uses of the hexasaccharides and to compositions containing them. Background to the Invention Many of the actions of growth factors on cells are 15 thought to be mediated by a family of inositol phosphoglycan (IPG) second messengers 5 . It is believed that the source of such IPGs is a "free" form of glycosyl phosphetidylinositol (GPI) present in cell membranes. IPGs are thought to be released by the action of 20 phophatidylinositol-specific phospholipases following ligation of growth factors to receptors on the cell surface. There is evidence that IPGs mediate the action of a large number of growth factors including insulin, nerve growth factor, hepatocyte growth factor, insulin 25 like growth factor I (IGF-I), fibroblast growth factor, transforming growth factor 5, the action of IL-2 on B cells and T-cells, ACTH signalling of adrenocortical cells, IgE, FSH and hCG stimulation of granulosa cells, thyrotropin stimulation of thyroid cells and cell 30 proliferation in the early developing ear and rat mammary gland. The family of IPG second messengers can be divided into two distinct sub-families, A-type and P-type, on the 35 basis of biological activity. The A-type modulate the activity of a number of insulin-dependent metabolic WO 00/39141 PCT/GB99/04400 2 effects such as acetylCoA carboxylase (activates), cAMP dependent protein kinase (inhibits), adenylate cyclase (inhibits) and cAMP phosphodiesterases (stimulates). In contrast, the P-type modulate the activity of enzymes 5 such as pyruvate dehydrogenase phosphatase (stimulates) and glycogen synthase phosphatase (stimulates). The A type mimic the lipogenic activity of insulin on adipocytes, whereas the P-type mimic the glycogenic activity of insulin on muscle'-'. 10 Soluble IPG fractions have been obtained from a variety of animal tissues including rat tissues (liver, kidney, muscle, brain, adipose and heart) and bovine liver. Until recently, however, it has not been possible to 15 isolate single purified components from tissue-derived IPG fractions, much less in sufficient quantities to allow structural characterisation. Accordingly, prior art studies have been largely based on the biological activities of the fractions, with only speculation, based 20 on indirect evidence from metabolic labelling and cleavage techniques, as to the identity of the active components. In W098/11116 and W098/11117, we describe the isolation 25 of active components of A-type and P-type IPG fractions from human liver and placental tissue. The biological activity of these isolates is confirmed, and certain aspects of their structure (for instance, mass spectrometry data) and properties are disclosed. A-type 30 substances are defined, for instance, as cyclitol containing carbohydrates which also contain Zn 2+ ions and optionally phosphate; P-type substances are said to be cyclitol-containing carbohydrates which also contain Mn 2 and/or Zn 2 ions and optionally phosphate. 35 WO 00/39141 PCT/GB99/04400 3 Other studies indicate that A-type IPGs are composed of myo-inositol, non-acetylated D-glucosamine, D-galactose and phosphate 6 ), and P-type of chiro-inositol, non acetylated D-galactosamine, D-mannose and phosphate 7 t. 5 We have also obtained, from large quantities of bovine liver, a partially purified glycolipid fraction that after treatment with bacterial phosphatidylinositol specific phospholipase C gave a water soluble fraction that inhibited cAMP dependent protein kinase"l. This 10 biologically active material could be partially sequenced and the results indicated the presence of a family of substances containing myo-inositol, non-acetylated D glucosamine, an undetermined hexose (either D-mannose or D-galactose), and a terminal N-acetyl-D-glucosamine 15 residue. In addition up to four a-D-galactopyranosyl units and up to three phosphate groups seemed to be present 10 1 . These partial data go some way towards determining the 20 chemical structure of the A-type IPGs, but still leave a considerable number of uncertainties. Nevertheless, it would be desirable to synthesise IPG analogues with activities at least partially mimicking those of the naturally occurring materials. To this end, we have 25 carried out the synthetic, structural and biological studies documented in the art 1 3 17 , as a result of which a number of basic sub-structures have been synthesised, their shapes and spectroscopic properties studied and aspects of their potential biological activity 30 investigated. For instance, we have synthesized inositol-containing disaccharides such as those referred to as compounds C3 (1-D-6-O-(2-amino-2-deoxy-a-D glucopyranosyl)-myo-inositol 1,2-(cyclic phosphate)) and C4 (lD-6-0-( 2 -amino-2-deoxy-a-D-glucopyranosyl)-chiro 35 inositol 1-phosphate) "), and demonstrated biological WO 00/39141 PCT/GB99/04400 4 activity, in the form of proliferative effects on the early developing inner ear of a chick embryo, for at least the myo-inositol-containing C3 1 . 5 Frick et all"] also disclose the synthesis of IPG analogues, both trisaccharides and hexasaccharides, which include mannose, glucosamine and inositol units. They conclude that a mannose side chain is necessary to maximise the insulin-mimetic activity of their products. 10 Summary of the Invention The present invention arises from the design and synthesis of novel- hexasaccharides of the general formula I: 15 R 0

O

2 YN RO ROR RaeO 0 O RO e ROb 0 XN RO OR 20 RO ORa lOR ROf 0 RO 0ORet 25 wherein: - each R is independently selected from hydrogen, an alkyl or substituted alkyl group, an acyl or substituted acyl group, a phosphate group (P03- or PO 3 H for instance) or a protecting group, or two of the R groups may be 30 cyclic phosphate; - NX group represents N 3 ; or in the NX group, X represents one or more groups independently selected from hydrogen, alkyl or substituted alkyl, acyl or substituted acyl, 35 - NY represents a phthalimido group (NPht) or N 3 ; or WO 00/39141 PCT/GB99/04400 5 in the NY group, Y represents one or more groups independently selected from hydrogen, alkyl or substituted alkyl, acyl or substituted acyl; or a salt or derivative of said compound. 5 Thus, in the compounds of formula I, the NX and NY groups may be phthalimido group (NPht) or N 3 , a secondary amine

(-NR

1 H), tertiary amine(-NRiR 2 ) or quaternary amine group

(-N*R

1

R

2

R

3 ), an amidino group (R 1

CONR

2 -) where X or Y is an 10 acyl or substituted acyl group. In these formulae, R 1 , R,, R 3 in the NX or NY groups are independently selected from hydrogen or C 1 -Cie alkyl or substituted alkyl, more preferably C 1

-C

5 alkyl. Preferred substituents include phthalimido group (NPht), N 3 , NH 3 * and AcHN. 15 Preferably, the compounds represented by formula I, contain no more than three phosphate groups. The constituent saccharide units in I are labelled a-f, 20 and in the following description the R groups are referred to according to their positions within each unit, e.g. R 2 , indicates the R group at position 2 in unit a. 25 Compound I contains the basic saccharide units believed to be present in A-type IPGs, i.e. a myo-inositol unit (a), a non-acetylated D-glucosamine unit (b), a mannose unit (c), a terminal N-acetyl-D-glucosamine residue (d) and D-galactose units (e) and (f). It also has a 30 reasonable structural overlap with the conserved linear glycan chain of the GPI anchors depicted as formula 2 in Figure 1. Thus, taking into account the immunological evidence that antibody probes generated against 2 cross react with IPGs from rat liver and block some of the 35 effects of insulin 1 2 ), compound I can reasonably be WO 00/39141 PCT/GB99/04400 6 expected to be useful as a synthetic analogue, or mimetic, of naturally-occurring A-type IPGs, and hence to have applications in pharmaceutical compositions and methods. 5 Other aspects of the invention relate to methods for synthesising compounds of formula I and to intermediate compounds generated during the syntheses. 10 Detailed Description Compounds of Formula I The first aspect of the invention provides a compound of formula I, as defined above, or a salt or other derivative thereof. 15 In formula I, where R is an alkyl or acyl groups, or substituted versions thereof (e.g. with halogeno, cyano, amino, carbonyl, and/or carboxy groups etc) it is preferably C 1 -Cl 0 , and more preferably C 1

-C

5 , and may be 20 primary, secondary or tertiary. The NX and NY groups may be phthalimido group (NPht) or

N

3 , a secondary amine (-NR 1 H), tertiary amine(-NRR 2 ) or quaternary amine group (-N*R 1

R

2

R

3 ), an amidino group 25 (RFCONR 2 -) where X or Y is an acyl or substituted acyl group. In these formulae, R 1 , R 2 , R 3 in the NX or NY groups are independently selected from hydrogen or C1-Cl alkyl or substituted alkyl, more preferably C1-C 5 alkyl. 30 As used herein, "salt" includes physiologically acceptable salts meaning pharmaceutically applicable or non-toxic salts. Such salts are formed by compounds of Formula I with acid groups, e.g. carboxyl, with alkali and alkaline earth metals such as Na, K, Mg and Ca, and 35 with physiologically acceptable organic amines, e.g.

WO 00/39141 PCT/GB99/04400 7 triethylamine and tris(2-hydroyethyl)amine. Compounds of Formula I containing basic groups, e.g. an amino group or guanidino groups (and especially NX or NY groups where X and/or Y are hydrogen) form salts with inorganic acids, 5 e.g. hydrochloric acids, sulphuric acid, acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid and p-toluenesulphonic acid. Compounds with equal numbers of acid and base groups can form internal salts and do not need a third salt component. 10 "Derivative" includes coordination complexes, for example with metal ions such as Zn". It also includes so-called "prodrug" forms of the compound, convertible either in vitro or in vivo into compounds of formula I. An example 15 of a suitable prodrug is a glycolipid derivative in which Ra is: -P-0 - diacyl glycerol, 0 20 which may be convertible to I following phospholipase cleavage. As set out above, the compounds of the invention may also include one or more groups for protecting hydroxyl or 25 phosphate groups. Phosphate protecting group such as phenyl, benzyl or hydroxypropylnitrile (Hoeben-Weyl, Methods of Organic Chemistry, volume 12/1 or 12/2, Teilheimer, Synthetic Methods of Organic Chemistry, Vol 45). Protecting groups for the OH of sugars including 30 benzyl, acetyl, benzoyl, pivaloyl, trityl, tert butyldimethylsilyl, benzylidene or isopropylidene. A second aspect of the invention provides a material (whether a compound or composition) which incorporates a 35 compound of formula I, chemically or physically bound to WO 00/39141 PCT/GB99/04400 8 a coupling partner such as a label, a supporting substrate, a carrier, an effector or inhibitor molecule or an immobiliser. 5 In I, suitable protecting groups for R include menthoxycarbonyl (MntCO), an acetal (in particular, two R groups may together represent a bridging acetal such as O-cyclohexylidene, 0-isopropylidene or O-benzylidene), tert-butyldimethylsilyl (TBDMS), benzyl (Bn), tert 10 butyldiphenylsilyl (TBDPS), etc... Many protecting groups suitable for use in the syntheses and reactions of saccharides are known and are well documented in standard reference works. The choice depends in part on the route by which the compound I is synthesised and/or on the uses 15 to which it is to be put, including perhaps on reactions which it is subsequently intended to undergo. Either or preferably both of R" and R'f are phosphate. R'a and R 2 , may together represent a cyclic phosphate group. 20 X is preferably H 3 . Y is preferably AcH. Preferred forms of compound I are those which are at least partially deprotected, i.e. in which one or more of the R groups is hydrogen. For instance, at least the R 25 groups in units a and b, more preferably units a, b and c, most preferably units a, b, c and d, are either hydrogen or phosphate (e.g. Rla is still preferably phosphate). 30 A particularly preferred form of compound I is that shown as formula 1 in Figure 1, in which all R groups are hydrogen, with the exception of Rla and R'M which are phosphate, X is H 3 ' and Y is AcH. 35 Preferred protected forms of I are those which have been WO 00/39141 PCT/GB99/04400 9 or could have been prepared by the synthetic methods also provided by this invention (see below). These methods place certain limitations on the nature of the protecting groups R, to ensure the correct stereochemistry of the 5 glycosidic linkages in I, namely: (a) R 2 c, R 2 e and R 2 are preferably permanent, non participating protecting groups; and (b) R" and R 6 are preferably temporary protecting groups chosen to permit orthogonal deprotection with 10 respect to all the permanent protecting groups in I. In addition, NX is preferably a non-participating group, whereas NY is preferably participating. 15 A participating group is one which participates in a glycosylation reaction and influences the stereochemistry of thQtglycosidic linkage formed, leading to a 1,2-trans linkage. A non-participating group is one which in principle does not influence the stereochemical outcome 20 of a glycosylation reaction. Preferred protecting groups for I include N 2 for X, Pht for Y and bridging acetals for the pairs R2a and R 3 ,, R" and R2a, R 4 d and R 6 d, R 3 * and R 4 * and R" and R 4 f. 25 A particularly preferred protected form of I is that in which R" is MntCO; R 2 ' and R 3 , together represent 0 cyclohexylidene, as do R 4 and R 5 a together; R", R", R 2 c,

R

4 c, R 3 d, R2e and R 2 are benzyl (Bn); R 4 d and R 6 d together 30 represent 0-benzylidene; R 3 " and R 4 *, and also R 3 f and R 4 , together represent O-isopropylidene; and R" is acetate. In some embodiments, the compounds and derivatives of the invention are be useful as IPG agonists, and more 35 preferably A-type IPG agonists, sharing one or more of WO 00/39141 PCT/GB99/04400 10 the biological properties of A-type IPGs. In other embodiments, the compounds and derivatives of the invention may act as IPG antagonists, and more preferably A-type IPG antagonists, e.g. by competing with the IPGs 5 and so reducing one or more IPG biological activities. A-type mediators modulate the activity of a number of insulin-dependent enzymes such as cAMP dependent protein kinase (inhibits), adenylate cyclase (inhibits) and cAMP 10 phospho-diesterases (stimulates). In contrast, P-type mediators modulate the activity of insulin-dependent enzymes such as pyruvate dehydrogenase phosphatase (stimulates) and glycogen synthase phosphatase (stimulates). The A-type mediators mimic the lipogenic 15 activity of insulin on adipocytes, whereas the P-type mediators mimic the glycogenic activity of insulin on muscle. Both A-and P-type mediators are mitogenic when added to fibroblasts in serum free media. The ability of the mediators to stimulate fibroblast proliferation is 20 enhanced if the cells are transfected with the EGF receptor. A-type mediators can stimulate cell proliferation in the chick cochleovestibular ganglia. Soluble IPG fractions having A-type and P-type activity 25 have been obtained from a variety of animal tissues including rat tissues (liver, kidney, muscle brain, adipose, heart) and bovine liver. A- and P-type IPG biological activity has also been detected in human liver and placenta, malaria parasitized RBC and mycobacteria. 30 The ability of an anti-inositolglycan antibody to inhibit insulin action on human placental cytotrophoblasts and BC3Hl myocytes or bovine-derived IPG action on rat diaphragm and chick cochleovestibular ganglia suggests cross-species conservation of many structural features. 35 However, it is important to note that although the prior WO 00/39141 PCT/GB99/04400 11 art includes these reports of A- and P-type IPG activity in some biological fractions, the purification or characterisation of the agents responsible for the activity is not disclosed. 5 A-type substances are cyclitol-containing carbohydrates, also containing Zn 2 * ion and optionally phosphate and having the properties of regulating lipogenic activity and inhibiting cAMP dependent protein kinase. They may 10 also inhibit adenylate cyclase, be mitogenic when added to EGF-transfected fibroblasts in serum free medium, and stimulate lipogenesis in adipocytes. P-type substances are cyclitol-containing carbohydrates, 15 also containing Mn 2 * and/or Zn 2 + ions and optionally phosphate and having the properties of regulating glycogen metabolism and activating pyruvate dehydrogenase phosphatase. They may also stimulate the activity of glycogen 'synthase phosphatase, be mitogenic when added to 20 fibroblasts in serum free medium, and stimulate pyruvate dehydrogenase phosphatase. Methods for obtaining A-type and P-type IPGs are set out in W098/11116 and W098/11117. 25 Pharmaceutical Compositions A third aspect of the invention provides a composition comprising a compound, derivative or material according to the first or second aspect (be they an IPG agonist or 30 antagonist), a pharmaceutically acceptable derivative thereof. The pharmaceutical composition may include other pharmaceutically acceptable adjuvants such as carriers, 35 buffers, stabilisers or other excipients, depending on WO 00/39141 PCT/GB99/04400 12 the purpose of the composition and its intended route of administration (e.g. oral, intravenous or whatever). It may additionally include other pharmaceutically active ingredients, which may be therapeutically (including 5 prophylactically) active or have some diagnostic function. It may for instance contain insulin, a P-type IPG or IPG analogue, another A-type IPG or analogue, and/or an IPG antagonist. The composition may also, of course, contain more than one compound, derivative or 10 material according to the first or second aspect of the invention. The composition may be in any suitable form, such as a tablet, capsule, powder or liquid for oral 15 administration, or a solution or suspension for use for instance as a vaccine. Conventional solid or liquid carriers may be used in such formulations. The concentration of the compound, derivative or material contained in the pharmaceutical composition will depend, 20 of course, on the nature and severity of the condition to be treated or diagnosed using the composition, and on the patient to whom and method by which it is to be administered. 25 Possible uses for the pharmaceutical composition of this third aspect of the invention (which include both therapeutic and diagnostic uses) are described below. Uses of the Compounds and Compositions 30 In preferred embodiment, the compounds of formula I are expected to mimic, at least to an -extent, the biological activity of A-type IPGs, they are equally expected to be of use in therapeutic and diagnostic methods based on that activity. Thus, fourth - sixth aspects of the 35 invention provide, respectively, a compound or derivative WO 00/39141 PCT/GB99/04400 13 according to the first aspect, or a material according to the second aspect, or an antagonist thereto, for use in any surgical, therapeutic or diagnostic method; the use of such a compound, derivative, material or antagonist in 5 the manufacture of a medicament for use in any surgical, therapeutic or diagnostic method; and a method of surgery, therapy or diagnosis which involves the use of such a compound, derivative, material or antagonist. 10 The term "therapy" as used here includes prophylaxis. Moreover, in this section "compound" should be taken to include derivatives, materials and antagonists as referred to in connection with the third aspect of the invention. 15 The compounds are in particular likely to be of use in treating and/or diagnosing any condition which is related to (ie, which is or can be caused or mediated, directly or indirectly, by, or which is in any way associated 20 with) insulin activity, in particular the effects of the IPG second messengers. They may be used, for instance, in the treatment and/or diagnosis of disorders in which the lipogenic response of a patient has in some way been affected so that he or she produces a relatively low 25 amount of A-type IPGs in response to growth factors such as insulin. More particularly, the compounds are likely to be of use in the treatment and/or diagnosis of diabetes, including 30 diabetes due to insulin resistance, insulin resistance in type I diabetes and brittle diabetes, and of conditions associated with insulin resistance or insulin underproduction, such as neurotrophic disorders or polycystic ovary disease. 35 WO 00/39141 PCT/GB99/04400 14 The use of both P- and A-type IPGs in the diagnosis and treatment of diabetes is disclosed in W098/11435. This application discloses that in some forms of diabetes the ratio of P:A-type IPGs.is imbalanced and can be corrected 5 by administering a medicament containing an appropriate ratio of P- or A-type IPGs or antagonist(s) thereof. In particular, it describes the treatment of obese type II diabetes (NIDDM) patients with a P-type IPG and/or an A type IPG antagonist and the treatment of IDDM or lean 10 type II diabetes (body mass index < 27) with a mixture of P- and A-type IPGs, typically in a P:A ratio of about 6:1 for males and 4:1 for females. The compounds and compositions of the present invention can be employed in such types of treatment. 15 The compounds of this invention are also likely to be of use in promoting either in vitro or in vivo neuron proliferation. They may thus have applications in the treatment and/or diagnosis of any condition related to 20 neuron proliferation. The neurons may be central (brain and spinal cord) neurons, peripheral (sympathetic, parasympathetic, sensory and enteric) neurons, or motor neurons. Treatments may involve the treatment of damage to the nervous system, of motor neuron disease, of 25 neurodegenerative disorders or of neuropathy. Damage to the nervous system includes the results of trauma, stroke, surgery, infection (e.g. by viral agents), ischemia, metabolic disease, toxic agents, or a combination of these or similar causes. Motor neuron 30 disease includes conditions involving spinal muscular atrophy, paralysis or amyotrophic lateral sclerosis. Neurodegenerative disorders include Parkinson's disease, Alzheimer's disease, epilepsy, multiple sclerosis, Huntingdon's chorea and Meniere's disease. 35 WO 00/39141 PCT/GB99/04400 15 A therapeutic treatment method in which the compounds may be used involves the administration to a patient suffering from a relevant condition a therapeutically (which includes prophylactically) effective amount of one 5 of the compounds, preferably in the form of a pharmaceutical composition according to the third aspect of the invention. "Effective amount" means an amount sufficient to cause a benefit (which may be prophylactic) to the subject or at least to cause a change in the 10 subject's condition. The actual amount administered to the patient, and the rate and time-course of administration, will depend on the nature of the subject, the nature and severity of the condition, the administration method used, etc.. Appropriate values can 15 be selected by the trained medical practitioner. The compound may be administered alone or in combination with other (treatments, either simultaneously or sequentially. It may be administered by any suitable route, including orally, intravenously, cutaneously, subcutaneously, 20 parenterally, nasally, intramuscularly, intraperitoneally, etc... It may be administered directly to a suitable site or in a manner in which it targets a particular site, such as a certain type of cell - suitable targeting methods are already known. 25 A diagnostic method according to the invention might involve the use of one of the compounds (which of course includes antagonists), or of a specific binding partner for it, or of a species which competes with it in binding 30 to another specific binding partner, to determine, either qualitatively or quantitatively, the existence of a particular medical condition or change in condition. Such a method may be carried out either in vitro or in vivo. One or more of the materials used in the method 35 may be appropriately labelled.

WO 00/39141 PCT/GB99/04400 16 A seventh aspect of the present invention provides a method of preparation of a pharmaceutical composition, involving admixing one or more of the compounds with one or more pharmaceutically acceptable adjuvants, and/or 5 with one or more other therapeutically active agents. Synthesis of the Compounds Further aspects of the present invention relate to the synthesis of compounds of formula I. The preferred 10 strategy allows their preparation from four readily available monosaccharide units, via three intermediate disaccharide "building blocks". These disaccharide intermediates have the general formulae II, III and IV: RO OR 6 f 15 OR R R R 4 bO 0 RO&f Rob RO RO 21 XN RO OR OR O SYNRO LR IIORO \eJ L IIIIIV R 2 eQ 20 in which R, X and Y have the same meanings as in formula I, and each L is a leaving group (or "activating group") which activates the anomeric position of the relevant saccharide unit in preparation for a glycosylation reaction with a glycosyl acceptor, or alternatively a 25 leaving group precursor (ie, a group which can be converted into a suitable leaving group). Each L must be chosen to ensure the optimum balance between reactivity at the anomeric position and selectivity in the unit as a whole, depending on the reactions which the unit in 30 question is to undergo. The constituent saccharide units of compounds II-IV are again labelled a-f, to reflect their correspondence with the units in formula I. 35 WO 00/39141 PCT/GB99/04400 17 In compound II, position 1 of unit a is ideally differentiated from the other positions of that unit by the choice of appropriate R groups. For instance, Rla may be MntCO and the rest of the unit may be protected as a 5 dicyclohexylidene acetal in which R" and R", and separately R" and R5a, together represent the bridging group O-cyclohexylidene. X is preferably N 2 . Rlb is conveniently H, or a removable precursor group such as TBDMS, in preparation for a subsequent glycosylation of 10 II. R 3 " and R 6 b may be protecting groups such as Bn. In compound III, L is preferably trichloroacetimidate (C(NH)CCl 3 ) or a leaving group precursor such as thiophenyl (SPh). SPh is a preferred leaving group 15 precursor because of its stability and its versatility, being readily convertible into a number of suitable leaving groups. R 2 ' needs to be a permanent, non participating group, preferably Bn. Suitable groups for

R

3 c and R' include Bn and TBDPS, preferably Bn for R 4 c and 20 TBDPS for R 3 c. R 3 c should preferably differ from R 2 c and

R

4 c. In unit d, NY must be a participating group, preferably NPht, and suitable protecting groups R include an acetal bridging group such as benzylidene acetal for positions 4 and 6, and Bn for position 3. 25 In compound IV, L is preferably trichloroacetimidate or a leaving group precursor such as thiophenyl. The R groups at positions 2, 3 and 4 of both units and position 6 of unit f are suitable protecting groups such as Bn, Ac or 30 (ideally for positions 3 and 4 in each unit) a bridging acetal such as 0-isopropylidene. However R" and R 2 must be permanent non-participating protecting groups such as Bn, and R 6 f must be a temporary protecting group chosen to permit orthogonal deprotection with respect to all 35 permanent protecting groups in the final product I.

WO 00/39141 PCT/GB99/04400 18 Accordingly, the eighth aspect of the present invention provides a method of synthesis of a compound of formula I, which involves the use, and preferably also the preparation, of at least two of the intermediate 5 disaccharide compounds II, III and IV. This method conveniently involves condensing together the at least two disaccharides to form a tetrasaccharide intermediate compound. The hexasaccharide product is preferably synthesised by reacting the tetrasaccharide intermediate 10 with a third disaccharide intermediate, preferably also selected from compounds II, III and IV. The method preferably involves reacting together at least compounds II and III, more preferably all three compounds II, III and IV. 15 The method optionally includes the removal, after formation of the hexasaccharide, of one or more protecting groups R, and/or their replacement with other groups such as phosphates, for instance to produce the 20 compound 1. Conventional chemical techniques may be used to effect such substituent changes, the nature and sequence of the reaction steps used depending on the nature of the R groups and on the groups with which they are to be replaced. 25 Preferably, compounds II and III are reacted together first, to form an intermediate tetrasaccharide of the general formula XI: 30RO YN OR RO -O RO OR 0 0 Ro Rt O OR 35 0 I WO 00/39141 PCT/GB99/04400 19 in which R, X and Y are as defined in connection with compounds II and III, and R 3 is preferably hydrogen. 5 Compound XI is then preferably glycosylated using compound IV as a glycosyl donor, to form compound I. This last step will involve the selective deprotection of compound XI at R3c to convert it into a glycosyl acceptor. 0 A ninth aspect of the invention therefore provides a method of synthesis of a compound of formula I, which involves the use, preferably also the preparation, of a tetrasaccharide intermediate of the formula XI and 5 preferably the reaction of that intermediate with a compound of formula IV. Compounds II, III and IV are preferably prepared from the monosaccharide "building blocks" represented by the 0 general formulae V-IX: RO OR RecO OR 2 C R OR RO L RO OR V RORO RL RO R 2 eO L R- uu RO 5 XN YN IX VI VIII in which R, X, Y and L have the same meanings as in formulae II-IV, the units again bearing letters corresponding to those which they will provide in ) compounds I-IV (although compound IX provides both units e and f in compound IV). Compound II is preferably prepared by firstly preparing the myo-inositol building block V and then glycosylating 5 using a D-glucosamine derivative such as VI as a glycosyl WO 00/39141 PCT/GB99/04400 20 donor. In the myo-inositol building block, position 6 should be free for glycosylation (i.e. R"6 should be hydrogen) and position 1 should ideally be differentiated, as explained in connection with compound 5 II. Compound V may be prepared from myo-inositol using a regioselective acylation reaction via a boron-tin exchange reaction 2

-

3 oj The glycosyl donor is preferably a 2-azido-2-deoxy-D 10 glucopyranosyl, ie, compound VI with X=N 2 . L is preferably a trichloroacetimidate leaving group, which may be precursed during the synthesis by for instance a thiophenyl group. Such a compound may be prepared, for instance, from a 2-amino-2-deoxy D-glucosamine 15 hydrochloride, via a diazo transfer reaction from trifluoromethanesulphonyl azidef" 3 . R b, Rb and Rb in the glycosyl donor may be any suitable protecting groups, such as Bn for R 3 b and R 6 b and TBDMS 20 (selectively removable) for R*. Generally speaking, R* should be different to R b and Rb, and more preferably R b and Rb are the same. Bridging acetals are not preferred as protecting groups since their subsequent reductive opening, during the glycosylation reaction, can lead to 25 hydrolysis of acetal protecting groups present on the myo-inositol unit. Since in compound II, R"l should ultimately be H, position 4b should be selectively deprotected following glycosylation of the myo-inositol block. 30 Compound III is preferably prepared by glycosylating the mannose derivative VII, with the glucosamine derivative VIII. In VII, L is ideally a leaving group precursor such as thiophenyl, which can be converted to a suitable 35 leaving group following glycosylation with VIII. R 6 c WO 00/39141 PCT/GB99/04400 21 should ultimately be hydrogen, leaving that position free for glycosylation. Suitable groups for R 2 c, R 3 ' and R" include Bn and TBDPS, preferably Bn for R 2 c and R 4 c and TBDPS for R 3 c. R 2 ' needs to be permanent and non 5 participating, as in III. R 3 c and R 6 c are mutually orthogonal temporary protecting groups chosen to allow deprotection without affecting the remaining groups at positions 1 and 4. 10 In the glucosamine derivative VIII used as the glycosyl donor, positions 3, 4 and 6 must be protected, whilst L must be a suitable leaving group, fluoride being preferred. Suitable protecting groups include an acetal bridging group such as benzylidene acetal for positions 4 15 and 6, and Bn for position 3. In the synthesis of the compounds, NY must be a participating group such as NPht. Compound IV is preferably prepared by reacting together a D-galactose-based glycosyl donor and acceptor, both 20 corresponding to formula IX, both of which can be prepared from appropriate D-galactose derivatives such as B-D-galactopyranose pentaacetate. In compound IX, for the glycosyl donor, L is a suitable leaving group such as trichloroacetimidate and R 2 ,, R 3 *, R" and R 6 ' represent 25 suitable protecting groups such as Bn, Ac or (ideally for positions 3 and 4) a bridging acetal such as 0 isopropylidene. For the glycosyl acceptor, R 6 should be hydrogen ready for glycosylation, whilst positions 2, 3 and 4 must be suitably protected such as with Bn or 30 (again conveniently for positions 3 and 4) a bridging acetal such as 0-isopropylidene. L must be a leaving group precursor, preferably a thiophenyl group. One limitation on the substituents is that R 2 e in both the glycosyl donor and acceptor must be a permanent non 35 participating group, Bn being preferred for both.

WO 00/39141 PCT/GB99/04400 22 Thus, the methods of the invention can be seen more preferably to involve preparing the intermediate compounds II, III and IV starting from the four monosaccharide units myo-inositol, D-glucosamine, D 5 mannose and -D-galactose, any of which may be in the form of a derivative such as a pentaacetate in which, for instance, one or more hydroxyl groups have been replaced by suitable protecting groups. The preferred four starting materials are: 10 HO OH OH HO OH AcO OAc HO- 0I~H HO HOI~ HO OH HO OH HO -OH AcOOAc

NH

2 AcO myo-inositol D-glucosamine D-mannose g-D-galactopyranose pentaacetate Accordingly, a tenth aspect of the invention provides a method of synthesis of a compound of formula I, which involves the use of myo-inositol, D-glucosamine, D mannose and S-D-galactose, and/or of suitable derivatives 20 thereof, as the basic monosaccharide starting materials. S-D-galactopyranose pentaacetate is preferably used in place of S-D-galactose itself. Clearly the protecting groups used during the synthesis 25 must be carefully chosen so as to ensure availability only of the appropriate substituents at any given time. Suitable groups are referred to above in connection with the compounds II-IX. 30 The substituents referred to as preferred in compounds V IX and/or in compounds II-IV are of course also preferred, in the corresponding positions, in the tetrasaccharide intermediate XI and in the final product I. 35 WO 00/39141 PCT/GB99/04400 23 Many of the intermediate compounds formed during a synthesis according to the invention are believed to be novel compounds. These include compounds 9, 10, 11, 12, 13, 18, 19, 20, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 5 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 48, 49 or 50 referred to in the Example below. In particular, an eleventh aspect of the invention provides a compound of the general formula II, or a salt or other derivative thereof, in which R" (which is preferably hydrogen, or a 10 protecting group chosen to permit orthogonal deprotection with respect to the other protecting groups present) is different to R lb and preferably also to R 6 b, and more preferably R" and R" are the same as each other but different to R*. 15 Twelfth to eighteenth aspects of the invention provide, respeditively, compounds of the general formulae III, IV, VI to IX and XI, as defined above, or in each case a salt or other derivative thereof. 20 A nineteenth aspect of the present invention provides a method of synthesis of a compound of formula II (as defined above), which involves firstly preparing a glycosyl donor, in the form of a 2-azido-2-deoxy-D 25 glucopyranosyl of formula VI (with X=N 2 ), from a 2-amino 2-deoxy D-glucosamine salt via a diazo transfer reaction from trifluoromethanesulphonyl azide (as shown in Figure 4), and then reacting that donor with a glycosyl acceptor in the form of a myo-inositol building block of formula 30 V. The method preferably also involves preparing compound V. The nature of the R and L groups are preferably as described above in connection with the preparation of intermediate II. In particular, R 4 b is preferably differentiated from the other R groups of the glycosyl 35 donor, so that in the product II position 4b is free for WO 00/39141 PCT/GB99/04400 24 further glycosylation, for instance for use in the synthesis of the tetrasaccharide XI and/or the hexasaccharide I. 5 Finally, a twentieth aspect of the invention provides a method of synthesis of a tetrasaccharide of the general formula XI, as defined above, by reacting together a glycosyl acceptor of formula II, in which R" is hydrogen, and a glycosyl donor of formula III, in which L is a 10 leaving group. The method preferably also involves preparing one or both of the compounds II and III; more preferably it involves preparing compound II in accordance with the nineteenth aspect of the invention. 15 Embodiments of the present invention will now be described in more detail by way of example and not limitation with reference to the accompanying drawings. Brief Description of the Drawings 20 Figure 1 shows the chemical formulae for (1) a preferred hexasaccharide according to the first aspect of the invention and (2) GPI anchors typically present in cell membranes. 25 Figure 2 shows a retrosynthetic analysis from which the methods of synthesis of the eighth to tenth aspects of the invention were derived; and Figures 3-9 illustrate reaction schemes for a method of 30 synthesis in accordance with the invention. Examples There is now described an exemplary synthesis, according to the present invention, of a compound of formula I. 35 The synthesis was devised using the retrosynthetic WO 00/39141 PCT/GB99/04400 25 analysis depicted in Figure 2. It proceeded via three intermediate disaccharide compounds shown as II, III and IV, which themselves could be prepared (via monosaccharide units V-IX) from the four starting 5 materials myo-inositol, D-glucosamine, D-mannose and S-D galactopyranose pentaacetate. Of note is the choice of protecting groups and leaving groups for the monosaccharide "building blocks" V-IX, to 10 ensure the selective reaction of appropriate substituents during each stage of the synthesis and the desired stereochemistry at each glycosidic link formed. Preparation of intermediate II 15 Figures 3-5 illustrate the preparation of disaccharide II from myo-inositol and D-glucosamine. A myo-inositol buildfig block 6 was prepared following a previously reported procedure 5 ' that is based on the well established regioselective enhancement of the 20 nucleophilicity of hydroxyl groups as tributyl tin ethers or dibutyl tin acetals 2 7 1, but overcoming the insolubility of myo-inositol in most organic solvents by using a boron-tin exchange reaction 8 3 01 (see also Potter et al1 2 11 for background on the preparation of myo-inositol 25 derivatives). Thus, myo-inositol was converted into the hexane soluble hexa-O-diethylboryl derivative 3 (100% yield), which was reacted in situ with dibutyl tin bis-acetylacetonate and 30 then with L-menthyl chloroformate to give a diastereomeric mixture of regioselectively monosubstituted derivatives 4 and 5, from which the desired diastereoisomer 4 could be separated. 4 was then transformed into the building block 6, leaving position 6 WO 00/39141 PCT/GB99/04400 26 free for glycosylation and position 1 differentiated, after protection as a dicyclohexylidene acetal. Cyclohexylidene acetals of myo-inositol have been 5 frequently used as intermediates in the preparation of glycosyl myo-inositols ' 3 . Compound 6 was most conveniently prepared using 1-ethoxycyclohexene for the acetalation reaction, in cyclohexanone under conditions of thermodynamic control. 10 The 1,2-cis glycosylation of 6 was conveniently carried out using a 2-azido-2-deoxy-D-glucopyranosyl trichloroacetimidate as the glycosyl donor. 2-azido-2 deoxy-glycosyl donors are currently employed in 15 oligosaccharide syntheses but most of the methods used for the preparation of the 2-azido-2-deoxy building blocks involve low diastereoselectivity and a large number of steps1 33

-

36 . There is however reported 3 7 ' a one pot synthesis of peracetylated 2-azido-2-deoxy sugars 20 from commercially available 2-amino-2-deoxy sugar hydrochlorides through a diazo transfer reaction from trifluoromethanesulphonyl azide, and this method can be used to prepare the glycosyl donors 15 and 17 as shown in Figure 4. 25 D-glucosamine hydrochloride was thus converted 371 into the tetra-O-acetylated 2-azido-2-deoxy derivative 8, which in turn was converted into the thioglycoside 9[381 that was then transformed using well established chemical 30 techniques [18,23,39-43] into the trichloroacetimidates 15 and 17, via the intermediates 10-14 and 10, 11 and 16, respectively (see "Materials and Methods"). Glycosylation of the myo-inositol building block 6 with WO 00/39141 PCT/GB99/04400 27 glycosyl donor 17 in the presence of trimethylsilyl triflate in diethyl ether( 231 afforded 18 as a 10:1 c/p3 mixture in 95% yield (Figure 5). The subsequent reductive opening of the benzylidene acetal" in this 5 mixture, however, resulted in partial hydrolysis of the cyclohexylidene acetals; the donor 15 was therefore preferred for the glycosylation to prepare intermediate compound II. 10 Thus, 15 was condensed with 6 under the conditions mentioned above, as shown in Figure 5, to give 19 (corresponding to intermediate II) as a 9:1 a/p mixture in 73% yield. Treatment of 19 with tetrabutyl ammonium fluoride 4 4 afforded the product 20 (with position 4 of 15 the glucosamine unit deprotected) in 83% yield. Preparation of intermediate III Referring now to Figure 6, compound III was prepared from the readily available mannose derivative, 1,6-anhydro-B 20 D-mannopyranose (21), via the protected mannose unit 29 which was then glycosylated with the glucosamine fluoride derivative 30. Conversion of 20 to 28 was carried out according to the method described in Klooterman et al 6 . Glycosylation of 29 was conveniently performed according 25 to the methodology reported in Suzuki", which gave with excellent yield and selectivity the disaccharide 31. This was then converted [18,19,23] into the trichloroacetimidate 33 (i.e. the intermediate III) via compound 32 (see "Materials and Methods"). 30 Preparation of intermediate IV Figure 7 illustrates the preparation of IV from the galactose derivative B-D-galactopyranose pentaacetate (34). This was converted [21,38,45] to the glycosyl acceptor WO 00/39141 PCT/GB99/04400 28 35, which was also further converted, via 36 and 37 (see "Materials and Methods"), into the glycosyl donor 38. The glycosylation reaction of 35 and 38 afforded the 5 disaccharide 39 as a 6:1 a/S mixture in 86% yield. This was further transformed[ 18

,

19

,

2 3 ) into the trichloroacetimidate 41 via 40 (see "Materials and Methods"). 41 corresponds to intermediate IV. 10 Combination of II, III and IV Figure 8 shows the preparation of the hexasaccharide product 44 corresponding to compound I. Firstly, disaccharides 20 (intermediate II) and 32 (intermediate III) were condensed together to give the tetrasaccharide 15 42, with excellent stereoselectivity and an 81% yield. A carefully controlled desilylation of 42 led with a good yield to the glycosyl acceptor 43 (corresponding to XI), which carries a hydroxyl group at position 3 of the D mannose unit. 20 43 was then glycosylated with the trichloroacetimidate 41 (intermediate IV) to give the hexasaccharide 44 as a 6.5:1 a/3 mixture in 83% yield. 25 Deprotection of 44 To reach the compound 1 from 44, conventional methods may be used to remove each of the protecting groups. As an example, firstly R" might be removed using an excess of LiOH in THF/MrOH at room temperature. This would 30 simultaneously remove the acetate group R 6 f, and also cause partial opening of the phthalimido group in unit d. The phthalimido group could be cyclised again by treatment with Et 3 N/Ac 2 0, which would lead to acetylation of both R" and R 6 f. The phthalimido group could then be WO 00/39141 PCT/GB99/04400 29 removed, for instance using a large excess of ethylenediamine in n-butanol at 90'C, with subsequent acetylation of the resulting amine under the usual conditions. O-deacetylation would then give a diol (i.e. 5 R" and R 6 f being H) which could be subjected to phosphorylation using the phosphoramidite procedure. Finally, treatment with hydrogen in the presence of 10% Pd/C would yield the final deprotected product 1. 10 Materials and Methods General Remarks: TLC was performed on precoated plates (Merck aluminium sheets silica 60 F 2

,

4 , Art. no. 5554); detection was effected by observation under UV light (254 nm), then visualised using sulfuric acid or 15 phosphomolybdic acid in EtOH followed by heating. Column chromatography was conducted with Silica Gel 60 (0.023 0.040 4m, E. Merck) using de flash procedure. Melting points were determined using a Reicher Jung Thermovar apparatus and are uncorrected. Specific rotations were 20 measured on a Perkin Elmer model 241 polarimeter. NMR spectra were recorded on Bruker AMX-200, Avance DRX-500, Varian Gemini-200, XL-300 or Unity 500 spectrometers. Chemical shifts are expressed in ppm and referred to the residual signal of the solvent used. Microanalysis was 25 carried out by the Analysis Department of the Instituto de Quimica Orgdnica General (CSIC) on a Heraus CHNO-Rapid apparatus. 2,3:4,5-Di-O-cyclohexylidene-1-0-(-)-menthoxycarbonyl-1D myo-inositol (6) and 2,3:5,6-di-O-cyclohexylidene-1-O-( 30 )-menthoxycarbonyl-1-D-myo-inositol (7). To a solution of 100 mg (0.276 mmol) of 1-0-(-) menthoxycarbonyl-myo inositol 26 1 (4) and 5.7 mg (0.03 mmol) of dried p-TsOH in 2 mL of cyclohexanone at room temperature was added 350 mL (2.76 mmol) of 1-ethoxycyclohexene. The reaction WO 00/39141 PCT/GB99/04400 30 mixture was stirred for 3 h 30 min, quenched with Et 3 N and evaporated. Silica-gel column chromatography (hexane EtOAc, 5:1) afforded 73 mg (50%) of 6 and 41 mg (28%) of 7. Compound 6: White solid. Rf (hexane-EtOAc, 4:1) = 5 0.26. Mp: 83-85*C. [oa]D-50.4 (c 1.0, CHC1 3 ). 'H NMR (CDC1 3 , 200 MHz) d: 0.76 (d, 3H, CH 3 Mnt), 0.88 (d, 3H, CH 3 Mnt), 0.92 (d, 3H, CH 3 Mnt), 1.00 - 1.13 (m, 1H, Mnt), 1.37 1.76 (m, 22H, 16H cyclohex, 6H Mnt), 1.92 - 2.00 (m, 1H, Mnt), 2.07 - 2.11 (m, 1H, Mnt), 2.70 (d, 1H, JOH,6 = 3.5 10 Hz, 1H, OH), 3.44 (dd, J 5 4 = 10.5 Hz, J,,6 = 9.0 Hz, 1H, H), 3.85 (dd, J 4

,

5 = 10.5 Hz, J 4 ,3 = 7.9 Hz, 1H, H 4 ), 4.10 4.14 (m, 1H, H 6 ), 4.33 (dd, J 3

,

2 = 6.2 Hz, J 3

,

4 = 7.9 Hz, 1H,

H

3 ), 4.54 (dt, 1H, Ment), 4.60 (dd, J 2 ,1= 4.5 Hz, J 2 ,3 = 6,1 Hz, 1H, H2), 4.79 (t, J 1

,

2 = J 1

,

6 = 4.6 Hz, 1H, H 1 ). 13 C NMR 15 (CDCl 3 , 50 MHz) d: 16.6, 21.2, 22.4 , 23.7, 24.0, 24.1, 24.3 , 25.4, 25.5, 26.5, 31.9, 32.1, 34.5, 35.0, 36.9, 37.0, 37.2, 41.1, 47.4, 72.5, 73.7, 76.6, 78.3 , 79.1, 79.6, 111.6, 113.3, 154.3. Compound 7: Colourless oil. Rf: 0.14 (hexane/EtOAc 4:1). 1 H NMR (CDCl 3 , 200 MHz): d 20 0.77 (d, 3H, CH 3 Mnt), 0.88 (d, 3H, CH 3 Ment), 0.91 (d, 3H, CH 3 Mnt), 1.00 - 1.15 (m, 1H, Mnt), 1.37 - 1.69 (m, 26H, 20H cyclohexylidene, 6H Mnt), 1.93 - 2.09 (m, 2H, Mnt), 2.95 (bs, 1H, OH), 3.40 (t, J 5

,

4 = J 5

,

6 = 10.1 Hz, 1H,

H

5 ), 3.88 (dd broad, J 4 5 = 10.6 Hz, J 4 ,3 = 6.5 Hz, 1H, H 4 ), 25 4.01 - 4.13 (m, 2H, H 6 , H 3 ), 4.58 (dt, 1H, Mnt), 4.70 (t,

J

2

,

1 = J 2

,

3 = 4.7 Hz, 1H, H 2 ), 4.89 (dd, J 1 ,2= 4.4 Hz, J 1

,

6 = 10.5 Hz, 1H, H 1 ). 1 3 C NMR (CDCl 3 , 50 MHz): d 16.8 (-), 21.1 (-), 22.4 (-), 23.9 (+), 24.0 (+), 24.4 (+), 25.4 (+), 26.7 (-), 31.9 (-), 34.5 (+), 35.2 (+), 36.8 (+), 36.9 30 (+), 38.2 (+), 41.2 (+), 47.6 (-), 74.5 (-), 74.6 (-), 74.7 (-), 75.3 (-), 78.4 (-), 79.3 (-), 81.8 (-), 111.4 (o), 114.0 (o), 154.5 (o). Phenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-D- WO 00/39141 PCT/GB99/04400 31 glucopyranoside (9). To a solution of 2.10 g (5.63 mmol) of 1,3,4,6-tetra-O-acetyl-2-azido-2-deOxy-D glucopyranose ("I in 45 mL of CH 2 C1 2 at room temperature was added 1.15 mL (11.25 mmol) of thiophenol and 3.12 mL 5 (25.31 mmol) of boron trifluoride diethyl etherate. The reaction mixture was stirred for 8 days, diluted with

CH

9 C1 2 , washed with NaCl and dried over Na 2

SO

4 . Silica-gel column chromatography (hexane-EtOAc, 3:1) afforded 1.58 g of 9 (66%), as a 3:1 a/S mixture, and 0.53 g of recovered 10 8 (25%). Rf (hexane-EtOAc, 3:1) = 0.27. 'H NMR for 9c, taken from the spectra of the a/I mixture, (200 MHz, CDC1 3 ) d: 1.96 (s, 3H, CH 3 CO), 1.99 (s, 3H, CH 3 CO), 2.04 (s, 3H, CH 3 CO), 3.96 (dd, Ja, 6 b = 12.4 Hz, J 6

,,

5 = 2.2 Hz, 1H, H 6 a), 4.02 (dd, J 2

,

3 = 10.6 Hz, J2, 1 = 5.7 Hz, 1H, H2), 15 4.23 (dd, J6b,6a = 12.4 Hz, J6b,5 = 5.1 Hz, 1H, Heb), 4.53 (ddd, J 5

,

4 = 10.2 Hz, J5,6b = 5.1 Hz, JS, 6 a = 2.2 Hz, 1H, H), 4.96 (+, J 4

,

3 = J = 10.1 Hz, 1H, H 4 ), 5.27 (dd, J 3 2 = 10.4 Hz, J 34 = 9.8 Hz, 1H, H 3 ), 5.58 (d, J 1

,

2 = 5.7 Hz, 1H, Hi), 7.,20 - 7.45 (m, 5H, ArH). 1 H NMR for 90, taken from 20 the spectra of the ac/S mixture, (200 MHz, CDCl 3 ) d: 1.94 (s, 3H, CH 3 CO), 1.97 (s, 3H, CH 3 CO), 2.02 (s, 3H, CH 3 CO), 3.34 (t, J 2

,

3 = 2,1 = 10.1 Hz, 1H, H2), 3.63 (ddd, J 5

,

4 = 9.8 Hz, J5,6b = 4.9 Hz, J 5 ,6 8 = 2.6 Hz, 1H, Hs), 3.92 - 4.21 (m, 2H, H 6 a, HEb), 4.42 (d, i 1

,

2 10.1 Hz, 1H, Hi), 4.86 25 (t, J 4

,

3 = 74, 5 = 9.7 Hz, 1H, H 4 ), 5.01 (t, 73, 2 = J = 9.7 Hz, 1H, H 3 ), 7.20 - 7.45 (m, 5H, ArH). Phenyl 2-azido 4,6-O-benzylidene-2-deoxy-1-thio-D glucopyranoside (10). To a solution of 3.00 g (7.09 30 mmol) of 9 in 110 mL of MeOH at room temperature was added 5 mL of sodium methoxide in MeOH (0.3M). After 20 min, the solution was neutralized with Amberlite IR-120, filtered and evaporated. The crude mixture of phenyl 2 azido-2-deoxy-1-thio-D-glucopyranosides obtained was WO 00/39141 PCT/GB99/04400 32 dissolved in 30 mL of CH3CN. 5.32 mL (35.45 mmol) of benzaldehyde dimethyl acetal and 67.4 mg (0.35 mmol) of p-toluensulfonic acid were added and the reaction mixture was stirred for 2 h at rt, quenched with Et3N and 5 evaporated. Silica-gel column chromatography (hexane EtOAc, 6:1) afforded 1.85 g of 10ay and 0.80 g of 10B (7:3 ratio, 97% total yield). Data for 10a.: white solid. Rf (hexane-EtOAc, 3:1) = 0.34. Mp: 127-128oC. [a]D+ 226.9 (c 1.09, CHCl3) . 'H NMR (200 MHz, CDCl3) d: 2.90 (d, JOH, 3 10 2.0 Hz, 1H, OH), 3.58 (t, J4,3 = Ja,5 = 9.3 Hz, 1H, H 4 ), 3.76 (t, J6a,5 - J6a,6b = 10.2 Hz, 1H, H 6 a), 3.92 (dd, J 2

,

3 9.8 Hz, J2,, = 5.4 Hz, 1H, H 2 ), 4.07 (dt, J,4 = J3,2 = 9.6 Hz, J3,O,= 2.0 Hz, 1H, H3), 4.24 (dd, J6b,6, = 10.2 Hz, J6b, = 4.9 Hz, 1H, Hsb), 4.41 (dt, J5,4 = J5,a = 10.2 Hz, JS6b = 4.9 15 Hz, 1H, H 5 ), 5.57 (s, 1H, H,), 5.58 (d, Ji,2 = 5.4 Hz, 1H, HI), 7.30 - 7.56 (m, 10H, ArH). "C NMR (50 MHz, CDC13) d: 63.46, 63.91, 68.51, 70.72, 81.68, 87.81, 102.18, 126.19, 126.29, 128.01, 128.40, 129.18, 129.42, 132.47, 133.05, 136.80. Anal. Calcd. for C, 9 Hi 9 N30 4 S: C, 59.21; H, 4.97; N, 20 10.90; S, 8.32. Found: C, 59.13; H, 5.08; N, 10.71; S, 8.13. Data for 100: white solid. Rf (hexane-EtOAc, 3:1) = 0.36. Mp: 152-154 0 C. [a]D - 65.8 (c 0.96, CHCl3). 'H NMR (200 MHz, CDCl3) d: 2.88 (d, JOH, 3 = 2.1 Hz, 1H, OH), 3.35 (dd, J2, 1 = 10.2, J 2 ,3 = 9.0 Hz, 1H, H2), 3.41 - 3.52 25 (m, 2H, H4, H), 3.73 (dt, J 3 ,2 = J3, 4 = 8.9 Hz, J3,OH, 2.1 Hz, 1H, H3), 3.77 (t, J6a,6b = J63,s = 10.2 Hz, 1H, H 6 ), 4.38 (dd, J 6 b, 6 a = 10.2 Hz, J6b,5 = 4.4 Hz, 1H, H 6 b), 4.52 (d, Ji,2 = 10.2 Hz, 1H, H,), 5.53 (s, 1H, H7), 7.35 - 7.61 (m, 10H, ArH). "C NMR (50 MHz, CDCl3) d: 65.20, 68.41, 70.27, 30 74.11, 80.22, 86.83, 101.94, 126.24, 128.38, .128.67, 129.12, 129.41, 130.88, 133.67, 136.74. Anal. Calcd. for C19Hj9,N0 4 S: C, 59.21; H, 4.97; N, 10.90; S, 8.32. Found: C, 59.09; H, 4.65; N, 10.81.

WO 00/39141 PCT/GB99/04400 33 Phenyl 2 -azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-1 thio-D-glucopyranoside (11). To a solution of 546 mg (1.42 mmol) of 100 in 9 mL of DMF at room temperature was added 43 mg (1.70 mmol) of sodium hydride and then 0.21 5 mL (2.84 mmol) of benzyl bromide. The reaction mixture was stirred for 40 min, quenched with a saturated aqueous solution of NaHCO 3 and dried over Na 2

SO

4 . Silica-gel column chromatography (hexane-EtOAc) afforded 110 in 98% yield. Following the same procedure, 11a was synthesized 10 using 10a as starting material in 95% yield. Data for 11p: white solid. Rf (hexane-EtOAc, 3:1) = 0.58. Mp: 106 108 0 C. [a]D - 121.0 (c 0.93, CHCl 3 ) .

1 H NMR (200 MHz, CDCl 3 ) d: 3.38 (dd, J 2

,

1 = 10.2, J 2

,

3 = 9.1 Hz, 1H, H 2 ), 3.45 (m, 1H, H), 3.60 - 3.69 (m, 2H, H 3 , H 4 ), 3.81 (t, 15 JEa, 6 b J a,5 = 10.2 Hz, 1H, Ha), 4.41 (dd, J 6 b, 6 a = 10.2 Hz, Job, = 4.9 Hz, 1H, H 6 b) , 4.51 (d, Ji, 2 = 10.2 Hz, 1H, H 1 ), 4.87 add, 2H, CH 2 Ph), 5.59 (s, 1H, H,), 7.31 - 7.61 (m, 15H, ArH). 13 C NMR (50 MHz, CDC13) d: 64.75, 68.50, 70.50, 75.19,,80.97, 81.31, 86.67, 101.29, 125.97, 128.00, 20 128.30, 128.42, 128.72, 129.11, 133.92, 137.09, 137.58. Anal. Calcd. for C 26

H

25

N

3 0 4 S: C, 65.67; H, 5.30; N, 8.84; S, 6.74. Found: C, 65.91 H, 5.21; N, 8.52; S, 6.58. Data for 11a: white solid. Rf (hexane-EtOAc, 3:1) = 0.54. Mp: 145 147 0 C. [a]D + 125.6 (c 0.74, CHC13). H NMR (200 MHz, 25 CDCl3) d: 3.73 - 3.83 (m, 1H, H 4 ), 3.78 (t, J 6 a, 6 b = J -a, = 10.3 Hz, 1H, H 6 a), 3.92 - 4.04 (m, 2H, H 2 , H 3 ), 4.24 (dd, 6b,63= 10.3 Hz, J 6 b,S = 5.0 Hz, 1H, Hsb), 4.44 (dt, J 5

,

4

J

5

,

6 a = 10.3 Hz, J5,6b = 5.0 Hz, 1H, H), 4.92 (dd, 2H,

CH

2 Ph), 5.58 (m, 1H, H 1 ), 5.62 (s, 1H, H7), 7.30 - 7.53 30 (m, 15H, ArH) .

3 C NMR (50 MHz, CDCl3) d: 63.61, 63.84, 68.60, 75.18, 77.82, 82.74, 87.90, 101.51, 126.01, 127.95, 128.23, 128.30, 128.42, 129.09, 129.17, 132.47, 133.01, 137.12, 137.67. Anal. Calcd. for C 26

H

25

N

3 0 4 S: C, 65.67; H, 5.30; N, 8.84; S, 6.74. Found: C, 65.50; H, WO 00/39141 PCT/GB99/04400 34 5.12; N, 8.68; S, 6.42. Phenyl 2-azido-3,6-di-O-benzyl-2-deoxy-1-thio-D glucopyranoside (12). A solution of 447 mg (0.94 mmol) 5 of 11P in 9.4 mL of THF containing 3A molecular sieves was stirred for 30 min at room temperature; after this, 1.201 g (18.16 mmol) of sodium cyanoborohydride was added. A saturated solution of hydrogen chloride in diethyl ether was then added dropwise until the evolution 10 of gas had ceased (pH < 7) and a TLC analysis showed conversion of all the starting material. The mixture was neutralized with a saturated aqueous solution of NaHCO 3 , diluted with CH 2 Cl2, filtered through celite, washed with water and dried over Na 2

SO

4 . Silica-gel column 15 chromatography (hexane-EtOAc, 3:1) afforded 430 mg of 12B (96%) as a colourless oil. Following the same procedure, 12a was synthesized using 11ar as starting material in 87% yield. Data for 123: Rf (hexane-EtOAc, 3:1) = 0.28. [a]D - 64.2 (c 1.10, CHC1 3 ). H NMR (200 MHz, CDC1 3 ) d: 2.70 20 (d, JO, 4 = 2.4 Hz, 1H, OH), 3.27 - 3.41 (m (ABX), 2H, H 2 ,

H

3 ), 3.47 (m, 1H, H 5 ), 3.65 (dt, J 4

,

3 = J4,5 = 8.5 Hz, J 4 ,s O= 2.4 Hz, 1H, H 4 ), 3.75 (dd, J6a,6b = 10.4 Hz, 6 3,5 = 4.3 Hz, 1H, Hea), 3.81 (dd, J 6 b,E = 10.4 Hz, JGb,S = 4.9 Hz, 1H, HEb), 4.45 (m (ABX), Ji, 2 = 9.9 Hz, 1H, H 1 ), 4.59 (dd, 2H, 25 CH 2 Ph), 4.87 (dd, 2H, CH 2 Ph), 7.28 - 7.61 (m, 15H, ArH). 1 3 C NMR (50 MHz, CDCl 3 ) d: 64.55, 70.27, 71.88, 73.72, 75.42, 78.05, 84.60, 86.23, 127.66, 127.82, 128.08, 128.17, 128.33, 128.45, 128.59, 128.96, 131.29, 133.48, 137.71, 137.86. Anal. Calcd. for C 26

H

2 7

N

3 0 4 S: C, 65.39; H, 30 5.70; N, 8.80; S, 6.71. Found: C, 65.61; H, 5.35; N, 8.58; S, 6.35. Data for 12a. Rf (hexane-EtOAc, 3:1) 0.31. [a]D + 124.9 (c 1.34, CHCl3). 'H NMR (200 MHz, CDCl 3 ) d: 2.51 (d, JOH,4 = 2.7 Hz, 1H, OH), 3.62 - 3.75 (m, 3H,

H

3 , H 6 a, H 6 b), 3.77 (dt, J 4

,

3 = J4,5 = 8.0 Hz, J 4 ,OH = 2.7 Hz, WO 00/39141 PCT/GB99/04400 35 1H, H4), 3.92 (dd, J 2

,

3 = 10.0 Hz, J 2

,

1 = 5.4 Hz, 1H, H 2 ), 4.35 (m, 1H, HO), 4.56 (dd, 2H, CH 2 Ph), 4.91 (dd, 2H,

CH

2 Ph), 5.58 (d, J 2

,

1 = 5.4 Hz, 1H, H 1 ), 7.25 - 7.54 (m, 15H, ArH). 13 C NMR (50 MHz, CDCl 3 ) d: 63.59, 69.72, 71.05, 5 72.36, 73.63, 75.39, 81.32, 87.28, 127.68, 127.80, 128.11, 128.17, 128.43, 128.65, 129.05, 132.16, 133.43, 137.71, 137.95. Anal. Calcd. for C 2 6

H

2 7

N

3 0 4 S: C, 65.39; H, 5.70; N, 8.80; S, 6.71. Found: C, 65.74; H, 6.05; N, 8.81; S, 6.60. 10 Phenyl 2-azido-3,6-di-O-benzyl-4-O-(tert butyldimethylsilyl)-2-deoxy-1-thio-D-glucopyranosi-de (13). A solution of 345 mg (0.72 mmol) of 123 and 287 mL (2.17 mmol) of collidine in 1 mL of CH 2 C1 2 was cooled at 15 00C. 249 mL (1.08 mmol) of tert-butyldimethylsilyl triflate were added dropwise during 2 h. The mixture was stirrdd for 10 min and quenched with water/ice, diluted and extracted with CH 2 C1 2 , washed with brine and dried over Na 2

SO

4 . Silica-gel column chromatography afforded 20 405 mg of 135 (95%) as a colorless oil. 13a was synthesized similarly using 12a as starting material in 98% yield. Data for 13B: Rf (hexane-EtOAc, 5:1) = 0.69. [a]D - 0.2 (c 0.65, CHC1 3 ). 'H NMR (200 MHz, CDC1 3 ) d: 0.01 (s, 3H, CH 3 ), 0.03 (s, 3H, CH 3 ), 0.88 (s, 9H, 25 'Bu), 3.24 - 3.40 (m (ABX), 2H, H 2 , H 3 ), 3.45 (m, 1H, H 5 ), 3.57 - 3.68 (m, 1H, H 4 ), 3.64 (dd, J6,,6b = 10.7 Hz, J 6 a,s = 5.4 Hz, 1H, H 6 a), 3.78 (dd, J6b, 6 a = 10.7 Hz, JEb,S = 2.1 Hz, 1H, Heb), 4.50 (m (ABX), J 1

,

2 = 9.7 Hz, 1H, H 1 ), 4.59 (dd, 2H, CH 2 Ph), 4.84 (dd, 2H, CH 2 Ph), 7.20 - 7.66 (m, 15H, 30 ArH). "C NMR (50 MHz, CDCl 3 ) d: -4.74, -3.78, 17.98, 25.91, 65.74, 69.12, 70.55, 73.37, 75.57, 80.70, 85.48, 86.49, 127.51, 127.59, 128.11, 128.33, 128.97, 131.71, 133.71, 137.98, 138.37. Anal. Calcd. for C 3 2

H

4 jN 3 0 4 SSi: C, 64.94; H, 6.98; N, 7.10; S, 5.42. Found: C, 65.45; H, WO 00/39141 PCT/GB99/04400 36 7.00; N, 6.96; S, 5.32. Data for 13a: Rf (hexane-EtOAc, 5:1) = 0.62. [a]D + 160.0 (c 1.38, CHCl 3 ). 1 H NMR (200 MHz, CDC1 3 ) d: 0.02 (s, 3H, CH 3 ), 0.05 (s, 3H, CH 3 ), 0.90 (s, 9H, 'Bu), 3.58 (dd, J 3

,

2 = 10.1 Hz, J 3 ,4 = 8.4 Hz, 1H, 5 H 3 ), 3.71 (bd, J = 3.8 Hz, 2H, H6a, H 6 ), 3.74 (Yt, J4,3 = 8.4 Hz, J 4

,

5 = 9.4 Hz, 1H, H 4 ), 3.95 (dd, J 2

,

3 = 10.1 Hz, J2,1 = 5.4 Hz, 1H, H2), 4.37 (dt, J 5

,

4 ='9.4 Hz, J, 6 3 = Js,6b = 3.8 Hz, 1H, H), 4.55 (dd, 2H, CH 2 Ph), 4.87 (dd, 2H,

CH

2 Ph), 5.63 (d, J 1

,

2 = 5.4 Hz, 1H, H 1 ), 7.21 - 7.61 (m, 10 15H, ArH). 13 C NMR (50 MHz, CDC1 3 ) d: -4.75, -3.71, 18.03, 25.94, 64.78, 68.91, 71.29, 73.13, 73.17, 75.20, 81.84, 87.41, 127.30, 127.47, 127.70, 128.27, 129.01, 132.44, 133.62, 138.06, 138.15. Anal. Calcd. for C 3 2

H

4 1

N

3 0 4 SSi: C, 64.94; H, 6.98; N, 7.10; S, 5.42. Found: C, 65.26; H, 15 6.77; N, 7.20; S, 5.50. 2-Azido-3,6-di-O-benzyl-4-O-(tert-butyldimethylsilyl)-2 deoxy-D-glucopyranose (14). A solution of 331 mg (0.56 mmol) of 130 in 12 mL of acetone was cooled to -15 *C in 20 darkness and 129 mg (0.73 mmol) of NBS were added. After 45 min, the reaction mixture was quenched with a saturated aqueous solution of NaHCO 3 , diluted and extracted with EtOAc, washed with brine and dried over Na 2

SO

4 . Silica-gel column chromatography (hexane-EtOAc, 25 7:1) afforded 279 mg of 14, as a 11:1 a/P mixture of anomers (quantitative yield). The same procedure was used for 13c to afford 14 in 91% yield. Rf (hexane-EtOAc, 6:1) = 0.15. M.p.: 76-78 0 C. 'H NMR for 14a (200 MHz, CDCl 3 ) d: -0.04 (s, 3H, CH 3 ), -0.03 (s, 3H, CH 3 ), 0.84 (s, 30 9H, tBu), 3.35 (dd, J 2

,

3 = 10.1 Hz, J 2

,

1 = 3.5 Hz, 1H, H 2 ), 3.49 (dd, J 6 a, 6b = 10.1 Hz, J6a,s = 6.9 Hz, 1H, H 6 a), 3.54 (dd, J 4

,

3 = 8.5 Hz, J 4

,

5 = 9.7 Hz, 1H, H 4 ), 3.69 (dd, J6b, 6a 10.1 Hz, J6b,5 = 2.1 Hz, 1H, H 6 b), 3.81 (dd, J 3

,

2 = 10.1 Hz, 73,4 = 8.5 Hz, 1H, H 3 ), 4.04 - 4.14 (m, 1H, Hs), 4.59 (dd, WO 00/39141 PCT/GB99/04400 37 2H, CH 2 Ph), 4.84 (dd, 2H, CH 2 Ph), 5.37 (bd, J = 3.2 Hz, 1H, H 1 ), 7.28 - 7.41 (m, 10H, ArH). 13 C NMR (50 MHz, CDCl 3 ) d: -4.81, -4.74, -3.75, 17.93, 25.85, 64.45, 67.78, 69.25, 71.16, 71.65, 71.83, 73.34, 73.47, 74.99, 76.07, 5 77.18, 80.11, 83.10, 92.05, 96.30, 127.38, 127.44, 127.66, 127.77, 127.91, 128.22, 128.42, 137.71, 138.15. Anal. Calcd. for C 96

H

37

N

3 0 5 Si: C, 62.50; H, 7.46; N, 8.41. Found: C, 62.80; H, 7.08; N, 8.15. 10 2-Azido-3,6-di-O-benzyl-4-O-(tert-butyldimethylsilyl)-2 deoxy-D-glucopyranosyl trichloraceti-midate (15). To a solution of 241 mg (0.48 mmol) of 14 in 2.5 mL of CH 2 Cl 2 at room temperature, were added 484 mL (4.83 mmol) of trichloracetonitrile and 67 mg (0.48 mmol) of flame dried 15 potassium carbonate. After 1 h 45 min, the reaction mixture was diluted with CH 2 C1 2 , filtered through celite and evaporated at reduced pressure. Silica-gel column chromatography (hexane-EtOAc, 10:1) afforded 198 mg of 15a and 85 mg of 150 (7:3 ratio, 91% total yield). Data 20 for 153. Rf (hexane-EtOAc, 6:1) = 0.43. [a]D + 28.5 (c 2.10, CHC1 3 ) .

1 H NMR (200 MHz, CDCl 3 ) d: 0.07 (s, 3H, CH 3 ), 0.09 (s, 3H, CH 3 ), 0.87 (s, 9H, 'Bu), 3.35 (dd, J = 9.6 Hz, J = 8.4 Hz, 1H, H 4 ), 3.54 - 3.83 (m, 4H, H 3 , H 5 , H 6 a, HEb), 3.69 (dd, J 2

,

3 = 10.6 Hz, J 2 ,1 = 8.3 Hz, 1H, H 2 ), 4.59 25 (dd, 2H, CH 2 Ph), 4.86 (dd, 2H, CH 2 Ph), 5.71 (d, J 1

,

2 = 8.3 Hz, 1H, H 1 ), 7.28 - 7.40 (m, 1OH, ArH), 8.80 (s, 1H, NH). 3 C NMR (50 MHz, CDCl 3 ) d: -4.82, -3.83, 18.00, 25.92, 66.13, 68.27, 70.28, 73.23, 75.07, 77.64, 83.42, 96.95, 127.36, 127.47, 127.54, 128.30, 138.15, 138.34, 161.01. 30 Data for 15a. Rf (hexane-EtOAc, 6:1) = 0.38. [a]D + 94.7 (c 1.38, CHC1 3 ). 1 H NMR (200 MHz, CDCl 3 ) d: 0.06 (s, 3H,

CH

3 ), 0.08 (s, 3H, CH 3 ), 0.90 (s, 9H, 'Bu), 3.65 - 3.95 (m, 6H, H 2 , H 3 , H 4 , H 5 , H 6 a, H 6 b), 4.57 (dd, 2H, CH 2 Ph), 4.89 (dd, 2H, CH 2 Ph), 6.50 (d, J 1

,

2 = 3.4 Hz, 1H, H 1 ), 7.32 - WO 00/39141 PCT/GB99/04400 38 7.41 (m, 10H, ArH), 8.75 (s, 1H, NH). "C NMR (50 MHz, CDCl 3 ) d: -4.83, -3.76, 18.00, 25.94, 63.68, 68.24, 70.48, 73.26, 74.90, 75.07, 80.38, 94.98, 127.34, 127.48, 128.22, 128.26, 137.90, 138.12, 160.81. Anal. Calcd. for 5 C 28

H

37 Cl 3

N

4 0 5 Si: C, 52.22; H, 5.79; N, 8.70. Found: C, 52.51; H, 5.45; N, 8.48. 2-Azido-3-O-benzyl-4, 6-O-benzylidene-2-deoxy-D glucopyranose (16). To a solution of 80 mg (0.168 mmol) 10 of 11a in 1.7 mL of acetone cooled at -15 'C in the dark was added 51.5 mg (0.289 mmol) of NBS. After stirring for 1 h 15 min, the reaction mixture was quenched with a saturated aqueous solution of NaHCO 3 , diluted and extracted with EtOAc, washed with brine and dried over 15 Na 2

SO

4 . Silica-gel column chromatography (hexane-EtOAc, 2:1), afforded 62 mg (96%) of 16 as a white solid, mixture of a/B (1:1) isomers. Rf (hexane-EtOAc, 2:1) 0.41. M.p.: 115-117 0 C. 'H NMR (CDCl 3 , 200 MHz) d: 3.08 (bd, JOH,1= 3.0 Hz, 1H, OH), 3.31 (dd, J 2

,

1 = 7.7 Hz, J 2

,

3 = 20 8.8 Hz, 1H, H2b), 3.30 - 3.37 (m, 1H, Hsb), 3.39 (dd, J 2

,

1 3.7 Hz, J 2

,

3 = 10.0 Hz, 1H, H2,), 3.51 (t, J4, 3 = J7,5 = 9.2 Hz, 1H, H 4 b), 3.60 - 3.73 (m, 4H, H 3 b, H 6 b, 2H), 3.98 4.07 (m, 2H, Ha), 4.20 (dd, J 6

,

5 = 4.9 Hz, J 6

,

6 = 10.3 Hz, 1H, HO), 4.24 (dd, J6, 5 = 5.0 Hz, J 6

,

6 '= 10.5 Hz, 1H, H 6 ), 25 4.50 (bdd, LOH = 3.1 Hz, J 1

,

2 = 7.8 Hz, 1H, Hlib), 4.78 (dd, 2H, CH 2 Phb), 4.80 (dd, 2H, CH 2 Ph.), 5.16 (bt, J 1

,

2 = J1,oH = 3.1 Hz, 1H, Hia), 5.49 (s, 1H, H 7 0), 5.51 (s, 1H, H 7 a), 7.16 - 7.44 (m, 10H, ArH) . 13C NMR (CDCl 3 , 50 MHz) d: 62.7, 63.5, 66.3, 67.2, 68.4, 68.9, 74.9, 75.1, 76.2, 79.0, 30 81.4, 82.7, 92.7 (C-la), 96.4 (C-lb), 101.3 (C-7), 101.4 (C-7), 125.9, 126.0, 127.9, 128.2, 128.3, 128.4, 129.1, 137.0, 137.1, 137.7. 2-Azido-3-O-benzyl-4, 6-O-benzylidene-2-deoxy-D- WO 00/39141 PCT/GB99/04400 39 glucopyranosyl trichloracetimidate (17). To a solution of 177 mg (0.46 mmol) of 16 in 2.5 mL of CH 2 Cl 2 at room temperature, were added 463 mL (4.62 mmol) of trichloracetonitrile and 64 mg (0.46 mmol) of activated 5 potassium carbonate. After 1 h 30 min, the reaction mixture was diluted with CH 2 C1 2 , filtered through celite and evaporated. Silica-gel column chromatography (hexane-EtOAc, 3:1) afforded 99 mg of 170 and 125 mg of a 1:5 mixture of 17a( and 173, respectively (92% total 10 yield, 1:10 a/S mixture). Data for 17. Rf (hexane EtOAc, 4:1) = 0.45. [a]D - 59.9 (c 0.99, CHCl 3 ) . 'H NMR (200 MHz, CDCl 3 ) d: 3.56-3.89 (m, 5H, H 2 , H 3 , H4, H 5 , H 6 .), 4.41 (dd, J 6

,

5 = 4.8 Hz, J 6 ,,, = 10.5 Hz, 1H, H), 4.90 (dd, 2H, CH 2 Ph), 5.60 (s, 1H, H,), 5.70 - 5.74 (m, 1H, Hj), 15 7.30 - 7.52 (m, 10H, ArH), 8.77 (s, 1H, NH) . "C NMR (CDCl 3 , 50 MHz) d: 65.5, 66.9, 68.3, 74.9, 79.0, 81.1, 96.7 (C-1), 101.4 (C-7), 125.9, 127.9, 128.1, 128.2, 128.3, 129.1, 136.9, 137.6, 160.8. Data for 17c. Rf (hexane-EtOAc, 4:1) = 0.36. 1 H NMR (200 MHz, CDCl 3 ) d: 20 3.60 - 3.88 (m, 3H), 4.00 - 4.12 (m, 1H, H), 4.19 (t, J = 9.5 Hz, 1H), 4.35 (dd, J 6

,

5 = 4.7 Hz, J 6

,

6 , = 10.2 Hz, 1H,

H

6 ), 4.94 (dd, 2H, CH 2 Ph), 5.63 (s, 1H, H 7 ), 6.38 (d, J 1 2 = 3.7 Hz, 1H, H 1 ), 7.31 - 7.51 (m, 10H, ArH), 8.75 (s, 1H, NH). 25 6-0-[2-Azido-3-0-benzyl-4,6-0-benzylidene-2-deoxy-ax-D glucopyranosyl]-2,3:4,5-di-O-cyclohexyliden-1-O menthoxycarbonyl-1D-myo-inositol (18a). A mixture of 56 mg (0.11 mmol) of 17, 23 mg (0.04 mmol) of 6 and 30 powdered 4A molecular sieves in 1.1 mL of ethyl ether was stirred for 45 min at room temperature. At this time, 76 mL (0.008 mmol) of a solution of trimethylsilyl triflate in ethyl ether (0.108M) were added dropwise. The reaction mixture was stirred for 15 min, quenched with WO 00/39141 PCT/GB99/04400 40 triethyl amine, diluted with CH 2 C1 2 , filtered through celite and evaporated in vacuo. Silica-gel column chromatography (hexane-EtOAc, 12:1) afforded 18 (37 mg, 95%) as a 10:1 a/S mixture of anomers. Data for 18ay: Rf 5 (hexane-EtOAc, 6:1) = 0.38. 'H NMR (500 MHz, CDCl 3 ) d: 0.77 (d, 3H, CH 3 Mnt), 0.88 (d, 3H, CH 3 Mnt), 0.92 (d, 3H,

CH

3 Mnt), 1.04 - 1.12 (m, 2H, Mnt), 1.27 - 1.74 (m, 25H, cyclohex, Mnt), 1.90 - 2.02 (m, 1H, Mnt), 2.10 - 2.18 (m, 1H, Mnt) , 3. 41 (dd, J2b, 3 b = 9. 8 Hz, J2blb = 3. 9 Hz, 1H, H2b) , 10 3.57 (dd, J 5 a, 4 a = 10.7 Hz, Jsa, 6 a= 8.8 Hz, 1H, H 5 a), 3.72 (Yt, J4b,3b= 9.3 Hz, J4b,5b = 9.8 Hz, 1H, H 4 b) , 3.74 (t, J6b,6b' = J6b,5b = 10.0 Hz, 1H, Hsb), 3.99 (dd, J 4

,

5 , = 10.7 Hz, J 4

,

3 , = 7.8 Hz, 1H, H 45 ), 4.05 (dd, J 6 a, 5= 8.8 Hz, J 6 a,a = 2.9 Hz, 1H, H 68 ), 4.07 (Yt, J3b,4b 9.3 Hz, J3b,2b = 9.8 Hz, 1H, Hab), 15 4.14 (dt, J5b,6b' = 4.9 Hz, JS, 6 b = 10.2 Hz, J5b, 4 b = 9.8 Hz, 1H, Hsb), 4.31 (dd, J6b', 6 b 10.0 Hz, J6b',sb = 5.1 Hz, 1H, HEb), q.39 (t, Jaa 2 = 3 3, 4 = 7.3 Hz, 1H, H 3 ), 4.50 - 4.55 (m, 1H, Mnt), 4.56 (dd, J 2 8 3, = 6.9 Hz, J 2 a,j,= 4.1 Hz, 1H,

H

2 a), 4,86 (dd, 2H, CH 2 Ph), 4.95 (Yt, Ja, 2 a= 3.9 Hz, Jla,6 = 20 2.9 Hz, 1H, Hia), 5.27 (d, Jlb, 2 b = 3.9 Hz, 1H, Hib), 5.57 (s, 1H, Hyb) , 7.25 - 7.35 (m, 10H, ArH) . 13C NMR (CDCl 3 , 50 MHz) d: 16.1, 20.7, 21.9, 23.2, 23.5, 23.7, 23.8, 24.9, 25.0, 26.0, 31.4, 34.0, 34.6, 36.2, 36.4, 36.6, 40.6, 47.0, 62.7, 62.8, 68.8, 73.1, 74.9, 76.2, 76.3, 76.4, 25 76.7, 79.0, 82.6, 97.5 (C-lb), 101.4 (C-7b), 112.1 (Cipso cyclohex), 113.3 (Cipso cyclohex), 125.9, 126.0, 127.8, 128.0, 128.2, 128.3, 128.4, 137.3, 137.9, 154.2. 6-0-[2-Azido-3,6-di-O-benzyl-4-0-(tert 30 butyldimethylsilyl) -2-deoxy-a-D-glucopyranosyl] -2,3:4,5 di-O-cyclohexylidene-1--menthoxycarbonyl-1D-myo-inositol (19a) . A mixture of 180 mg (0.28 mmol) of 15P, 73 mg (0.14 mmol) of 6 and powdered 4A molecular sieves in 3 mL of ethyl ether was stirred for 45 min at room WO 00/39141 PCT/GB99/04400 41 temperature. At this time, 194 mL (0.02 mmol) of a solution of trimethylsilyl triflate in ethyl ether (0.108M) were added dropwise over 45 min. The reaction mixture was stirred for 15 min, quenched with triethyl 5 amine, diluted with CH 2 C1 2 , filtered through celite and evaporated in vacuo. Silica-gel column chromatography (hexane-EtOAc) afforded 19 (102 mg, 73%) as a 9:1 a/B mixture of anomers. Data for 19a. Rf (hexane-EtOAc, 3:1) = 0.76. M.p.: 72-74'C. [a] D + 47.2 (c 1.36, CHCl3). 10 1 H NMR (500 MHz, CDCl 3 ) d: -0.03 (s, 3H, CH 3 Si), 0.02 (s, 3H, CH 3 Si), 0.76 (d, 3H, Mnt), 0.86 (s, 9H, 'Bu), 0.87 (d, 3H, CH 3 Mnt), 0.92 (d, 3H, CH 3 Mnt), 1.00-1.10 (m, 2H, Mnt), 1.20 - 1.70 (m, 25H, cyclohex., Mnt), 1.90 - 1.97 (m, 1H, Mnt), 2.07 - 2.13 (m, 1H, Mnt), 3.32 (dd, J2b, 3 b = 9.8 Hz, 15 J2b,ib = 3.4 Hz, 1H, H 2 b), 3.59 (dd, J5a,4a = 10.7 Hz, J 5 a, 6 , = 8.8 Hz, 1H, H 5 a), 3.64 (dd, J6b, 6 ,' = 10.9 Hz, J 6 b,5b = 1.9 Hz, 1H, H 6 b), 3.72 (dd, J6b',6b = 10.9 Hz, J6b,5b = 3.7 Hz, 1H, H 6 b.), 3.76 (Yt, J3b,4b = J3b,2b = 9.8 Hz, 1H, H 3 b), 3.81 (Yt, J4b,3b = J4b,5b = 9.8 Hz, 1H, H 4 b), 3.98 (dd, J 4 a,sa = 10.7 20 Hz, J 4 ,,3, = 7.3 Hz, 1H, H 4 ,), 3.98 - 4.02 (m, 1H, H 5 b), 4.14 (dd, J 6 a,5a = 8.8 Hz, J 6 .,1, = 3.4 Hz, 1H, H 6 3) , 4.37 (t, J3., 2 , = 3,41 = 7.3 Hz, 1H, H 3 a), 4.52 (dt, 1H, Mnt), 4.55 (dd, 2H, CH 2 Ph), 4.58 (dd, J 2 3, 3 = 7.3 Hz, J 2 a,a = 3.4 Hz, 1H, H 2 3), 4.82 (dd, 2H, CH 2 Ph), 5.00 (t, Ja, 2 a = Ja,6a = 3.4 25 Hz, 1H, Ha), 5.31 (d, Jib,2b = 3.4 Hz, 1H, Hib), 7.25 - 7.35 (m, 10H, ArH). 13 C NMR (50 MHz, CDCl 3 ) d: -4.95, -3.65, 16.13, 18.04, 20.76, 21.92, 23.22, 23.52, 23.63, 23.83, 23.90, 24.90, 25.06, 25.92, 31.43, 34.09, 34.53, 36.33, 36.65, 40.62, 47.01, 63.46, 68.40, 70.66, 72.14, 73.26, 30 74.51, 76.21, 76.55, 76.71, 77.18, 79.21, 80.39, 96.80, 112.06, 113.23, 127.30, 127.42, 128.24, 138.26, 154.14. Anal. Calcd. for C 55

H

81

N

3 0 12 Si: C, 65.77; H, 8.13; N, 4.28. Found: C, 65.72; H, 8.40; N, 4.28.

WO 00/39141 PCT/GB99/04400 42 6-0-(2-Azido-3,6-di-O-benzyl-2-deoxy-a-D-glucopyranosyl) 2,3:4, 5 -di-O-cyclohexylidene-1-O-menthoxycarbonyl-1D-Myo inositol (20a). To a solution of 82 mg (0.08 mmol) of 19c in 0.8 mL of THF were added 204 mL (0.24 mmol) of a 5 1M solution of tetrabutylammonium fluoride in THF. The reaction mixture was stirred for 45 min, quenched with water, diluted and extracted with CH 2 C1 2 and washed with brine. Silica-gel column chromatography (hexane-EtOAc, 6:1) afforded 20ay (61 mg, 84%). Rf (hexane-EtOAc, 3:1) = 10 0.46. M.p.: 74-76 0 C. [aoD + 25.9 (c 0.99, CHCl 3 ). 'H NMR (200 MHz, CDCl 3 ) d: 0.77 (d, 3H, Mnt), 0.89 (d, 3H,

CH

3 Mnt), 0.93 (d, 3H, CH 3 Mnt), 0.99 - 1.17 (m, 2H, Mnt), 1.23 - 1.75 (m, 25H, cyclohex., Mnt), 1.87 - 2.05 (m, 1H, Mnt), 2.07 - 2.20 (m, 1H, Mnt), 2.75 (d, JOH, 4 b= 2.2 Hz, 15 1H, OH), 3.35 (dd, J2b,3b = 9-9 Hz, J2b,1b = 3.6 Hz, 1H, H2b), 3.57 (dd, J 5 ,4 = 10.8 Hz, Jsa, 6 a = 8.5 Hz, 1H, H 5 s), 3.67 (dd, -6b,b' = 10.1 Hz, J6b,5b = 4.9 Hz, 1H, HEb), 3.77 - 3.86 (m, 3H), 4.00 (dd, J 4 a, 5 a = 10.8 Hz, J4,, 3 , = 7.4 Hz, 1H,

H

48 ), 4,,01 - 4.09 (m, 1H), 4.06 (dd, JEa,so = 8.5 Hz, J 6

,

1 = 20 2.6 Hz, 1H, H 6 a) , 4.40 (t, J 3 a, 4 a = J 3 a, 2 a = 7.2 Hz, 1H, H 3 3), 4.46 - 4.60 (m, 2H, H 2 ,, Mnt), 4.58 (dd, 2H, CH 2 Ph), 4.89 (dd, 2H, CH 2 Ph), 4.99 (dd, Jia,2a = 3.9 Hz, Jla,Eo = 2.6 Hz, 1H, Hia), 5.26 (d, J1b,2b = 3.6 Hz, 1H, Hib), 7.29 - 7.45 (m, 10H, ArH). 11C NMR (50 MHz, CDCl 3 ) d: 16.12, 20.73, 21.92, 25 23.22, 23.50, 23.65, 23.81, 23.88, 24.94, 25.04, 25.98, 31.42, 34.07, 34.55, 36.23, 36.49, 36.57, 40.61, 46.98, 62.50, 69.67, 70.50, 72.85, 73.16, 73.74, 76.19, 76.55, 76.70, 79.23, 79.72, 97.15, 112.12, 113.24, 127.66, 127.83, 127.90, 128.00, 128.44, 128.53, 137.69, 138.21, 30 154.16. Anal. Calcd. for C 4 9

H

67

N

3 0 1 2 : C, 66.12; H, 7.59; N, 4.72. Found: C, 65.91; H, 7.67; N, 4.52. 1,6-Anhydro-3-O-(4-methoxybenzyl)-B-D-mannopyranose (23). To a solution of 3.5 g (12.5 mmol) of 1,6-anhydro-2,3-0- WO 00/39141 PCT/GB99/04400 43 endo-(4-methoxybenzyliden)-S-D-mannopyranose(3 6 1 in 125 mL de CH 2 Cl 2 at 0 0 C was added slowly 40 mL (40 mmol) of a solution of DIBALH in toluene (lM). After 5 h, Et 3 N and MeOH were added. The crude reaction mixture was diluted 5 with EtOAc, washed with a solution of HCl (10%) and extracted with EtOAc. The organic layers were evaporated and the crude was purified by silica-gel column chromatography (CH 2 Cl 2 -MeOH, 20:1) affording 2.8 g (79%) of 23. Rf (CH2Cl 2 -MeOH 20:1) = 0.16. M.p. 108-110'C. 10 [ao]D: - 66.8 (c 0.72, CHC1 3 ). 'H RMN (acetone, 200 MHz) d: 2.88 (s, 1H, OH), 3.33 (d, 1H, OH), 3,56 (bt, 1H, H-6), 3.62 - 3.68 (m, 1H, H2), 3.78 (s, 3H, CH30), 3.92 (d, 1H, H3), 4.08 (d, 1H, H 6 .), 4.27 (d, 1H, H 4 ), 4.40 (d, 1H, H1), 4.56 (dd, 2H, CH2Ph), 5.12 (bs, 1H, HI), 6.91 (d, 2H, 15 ArH), 7.31 (d, 2H, ArH). 1 3 C NMR (CDCl, 50 MHz) d: 55.1, 64.5, 65.8, 69.0, 73.4, 75.7, 78.0, 101.8 (C-1), 129.2, 129.3, 129.5, 158.4. 1,6-Anhydro-2,4-di-O-benzyl-3-O-(4-methoxybenzyl)-B-D 20 mannopiranose (24). To a solution of 2.4 g (8.5 mmol) of 23 in 20 mL of DMF at room temperature were added 472 mg (18.7 mmol) of NaH and 1.9 mL (25.5 mmol) of BnBr. After 2 h, MeOH was added and the reaction mixture was diluted with EtOAc, washed with H20, dried over Na2SO 4 and 25 evaporated. Silica-gel column chromatography (hexano/EtOAc 3:1) afforded 3.9 g (quantitative yield) of 24. Rf (hexane-EtOAc 2:1) = 0.31. M.p. 68-70 0 C. [a]jD: 20.3 (c 0.84, CHCl 3 ) . 'H RMN (CDCl3, 200 MHz) d: 3.48 (bt, 1H, H 4 ), 3.60 (dd, J2,1= 1.7 Hz, J2, 3 = 5.3 Hz, 1H, H2), 30 3.66 (dd, J 6

,

6 = 7.0 Hz, J 6 ,, = 6.0 Hz, 1H, H), 3.74 (s, 3H, CH 3 0), 3.74 - 3.80 (m, 1H, H3), 4.18 (dd, J 6

,

5 = 0.9 Hz, J 6 ,6 = 7.1 Hz, 1H, H6'), 4.35 - 4.56 (m, 7H, H5, 3 CH2Ph), 5.41 (bt, 1H, H1), 6.89 (d, 2H, ArH), 7.26 - 7.40 (m, 12H, ArH). "C NMR (CDCl, 50 MHz) d: 55.1, 64.8, WO 00/39141 PCT/GB99/04400 44 71.1, 71.2, 72.7, 73.9, 74.4, 76.4, 100.0 (C-1), 127.5, 127.6, 127.8, 128.2, 128.3, 129.7, 137.6, 137.9, 159.2. 1,6-di-O-Acetyl-2,4-di-O-benzyl-3-O-(4-methoxybenzyl)-a 5 D-mannopyranose (25). A solution of 4.88 g (10.55 mmol) of 24 and 240 mL (1.24 mmol) of trimethylsilyltrifluoromethanesulphonate in 33 mL of acetic anhydride was stirred for 1 h at 0 0 C and 2 h at room temperature. The reaction mixture was diluted with 10 EtOAc, carefully washed with a saturated aqueous solution of NaHCO 3 , extracted with EtOAc and dried over Na 2

SO

4 . Silica-gel column chromatography afforded 25a (4.72 g, 79%) and 245 (169 mg, 3%). Data for 24ay: Rf (hexane EtOAc 2:1) = 0.36. [a]lD + 28.1 (c 0.78, CHC13). 'H NMR 15 (200 MHz, CDC1 3 ) d: 2.04 (s, 3H, CH 3 CO), 2.05 (s, 3H,

CH

3 CO), 3.72 (Yt, J 2 ,1 = ' 2

,

3 = 2.4 Hz, 1H, H 2 ), 3.81 (s, 3H, CH3O), 3.82 - 4.03 (m, 3H, H 3 , H4, H 5 ), 4.30 - 4.33 (m 2H, H6a, H 6 b), 4.54 (s, 2H, CH 2 Ph), 4.75 (dd, 2H, CH 2 Ph), 4.77 (dd, 2H, CH 2 Ph), 6.18 (d, J, 2 = 2.1 Hz, 1H, H 1 ), 6.83 20 - 7.40 (m, 14H, ArH) . "C NMR (50 MHz, CDCl 3 ) d: 20.79, 20.91, 55.24, 63.17, 71.71, 72.39, 73.38, 73.80, 75.25, 78.77, 91.65, 113.80, 113.95, 127.78, 127.86, 128.11, 128.35, 128.42, 129.37, 130.04, 137.78, 138.00. Anal. Calcd. for C 32

H

36 0 9 : C, 68.08; H, 6.43. Found: C, 68.29; H, 25 6.12. Data for 24B: Rf (hexane-EtOAc, 2:1) = 0.31. [o]D + 0.7 (c 4.22, CHCl 3 ). 1 H NMR (200 MHz, CDCl 3 ) d: 2.05 (s, 3H, CH 3 CO), 2.09 (s, 3H, CH 3 CO), 3.55 - 3.66 (m, 1H, H), 3.63 (dd, J 3

,

2 = 2.8 Hz, J 3

,

4 = 9.1 Hz, 1H, H 3 ), 3.82 (s, 3H, CH 3 0), 3.87 - 3.96 (m, 2H, H 2 , H 4 ), 4.30 - 4.35 (m 2H, 30 H 6 a, H 6 b), 4.57 (dd, 2H, CH 2 Ph), 4.76 (dd, 2H, CH 2 Ph), 4.87 (s, 2H, CH 2 Ph), 5.60 (d, J, 2 = 0.9- Hz, 1H, HI), 6.84 7.48 (m, 14H, ArH) . "C NMR (50 MHz, CDCl 3 ) d: 14.10, 20.80, 20.93, 55.16, 60.27, 63.25, 71.78, 73.37, 73.81, 74.07, 74.36, 75.03, 81.75, 92.92, 113.81, 127.61, WO 00/39141 PCT/GB99/04400 45 127.79, 128.00, 128.10, 128.14, 128.35, 129.24, 129.76, 137.83, 138.16, 159.28, 168.8, 170.74. Anal. Calcd. for C32H3609: C, 68.08; H, 6.43. Found: C, 67.84; H, 6.71. 5 1,6-di-O-Acetyl-2, 4 -di-O-benzyl-a-D-mannopyranose (26). To a solution of 100 mg (0.18 mmol) of 25 in 1.5 mL of

CH

2 Cl 2 was added 50 mL of trifluoroacetic acid in 2 mL of

CH

2 Cl 2 . The reaction mixture was stirred for 3 h at room temperature, neutralized with a saturated aqueous 10 solution of NaHCO 3 , extracted with CH 2 C1 2 and dried over Na 2

SO

4 . Silica-gel column chromatography (hexane-EtOAc, 3:1) afforded 26 (77 mg, 98%). Rf (hexane-EtOAc, 2:1) = 0.24. [a], + 29.7 (c 1.52, CHC1). 'H NMR (200 MHz, CDCl 3 ) d: 2.08 (s, 3H, CH 3 CO), 2.09 (s, 3H, CH 3 CO), 2.43 (d, JO, 3 15 = 9.7 Hz, 1H, OH), 3.68 (t, J 4

,

3 = J4,5 = 9.6 Hz, 1H, H 4 ), 3.74 (dd, J 2

,

3 = 3.8 Hz, J 2

,

1 = 1.8 Hz, 1H, H2), 3.88 (dd., J5,4 = 9.8 Hz, JS,,, = 4.6 Hz, JS 6 b = 2.3 Hz, 1H, H), 4.01 (dt, J 3

,

4 = J 3 ,OH = 9.6 Hz, J 3

,

2 = 3.8 Hz, 1H, H 3 ), 4.30 (dd, J6,,6b = 12.0 Hz, J 6 a,, = 4.6 Hz, 1H, H 6 a), 4.38 (dd, J76b,6 = 20 12.0 Hz, JEb,S = 2.3 Hz, 1H, H 6 b), 4.71 (dd, 2H, CH 2 Ph), 4.78 (dd, 2H, CH 2 Ph), 6.27 (d, J 1

,

2 = 1.8 Hz, 1H, HI), 7.30-7.41 (m, 10H, ArH). 13C NMR (50 MHz, CDCl 3 ) d: 20.80, 20.91, 63.14, 71.47, 71.62, 72.67, 75.05, 75.63, 76.78, 90.73, 127.98, 128.11, 128.25, 128.48, 128.64, 128.64, 25 137.15, 137.91, 168.91, 170.74. Anal. Calcd. for C 2 4

H

2 8 0 8 : C, 64.86; H, 6.35. Found: C, 64.44; H, 6.38. 1,6-di-O-Acetyl-2, 4-di-O-benzyl-3-O-(tert butyldiphenylsilyl)-aY-D-mannopyranose (27). To a solution 30 of 1.40 g (3.15 mmol) of 26, 170 mg (1.39 mmol) of 4 dimethylaminopyridine and 857 mg (12.60 mmol) of imidazole in 5 mL of DMF, were added 1.64 mL (6.30 mmol) of tert-butyldiphenylsilyl chloride. The reaction mixture was stirred for 17 h at room temperature, diluted WO 00/39141 PCT/GB99/04400 46 with ethyl ether, washed with water and brine and dried over Na 2

SO

4 . Silica-gel column chromatography (hexane EtOAc, 10:1, 4:1) afforded 27 (1.91 g, 89%). Rf (hexane EtOAc, 2:1) = 0.55. M.p. = 107-109'C [a]D + 44.8 (c 1.13, 5 CHC1 3 ) .

1 H NMR (200 MHz, CDCl 3 ) d: 1.14 (s, 9H, 'Bu), 1.89 (s, 3H, CH3CO), 2.04 (s, 3H, CH3CO), 3.03 (bs, 1H, H 2 ), 3.85 (ddd, J 5

,

4 = 9.6 Hz, JS 6 , = 4.3 Hz, J5,Sb = 2.2 Hz, 1H,

H

5 ), 4.07 (bt, J 4

,

3 = J4,5 = 9.2 Hz, 1H, H 4 ), 4.22 - 4.37 (m, 2H, H 6 a, H 6 b), 4.32 (dd, J 3

,

4 = 8.9 Hz, J 3

,

2 = 3.1 Hz, 10 1H, H3), 4.42 (dd, 2H, CH 2 Ph), 4.58 - 4.72 (bm, 1H,

CH

2 Ph), 4.99 - 5.12 (bm, 1H, CH 2 Ph), 5.95 (d, J 1

,

2 = 2.1 Hz, 1H, HI), 7.24 - 7.77 (m, 20H, ArH). 13 C NMR (50 MHz, CDCl 3 ) d: 19.32, 20.81, 27.06, 63.22, 72.05, 72.69, 72.99, 75.23, 76.36, 77.05, 91.30, 127.21, 127.31, 127.46, 15 127.66, 127.72, 127.77, 128.00, 128.18, 128.38, 129.83, 130.03, 133.12, 134.13, 135.92, 136.08, 137.86, 138.16. Anal. falcd. for C 40

H

46

C

8 Si: C, 70.36; H, 6.79. Found: C, 70.61; H, 6.77. 20 Phenyl 6-O-acetyl-2, 4-di-O-benzyl-3-O-(tert butyldiphenylsilyl)-l-thio-aY-D-mannopyranoside (28a). To a solution of 1.80 g (2.64 mmol) of 27 in 26 mL of CH 2 C1 2 at room temperature were added 592 mL (4.80 mmol) of thiophenol and 1.32 mL (10.5 mmol) of borontrifluoride 25 diethyl etherate. The reaction mixture was stirred for 30 min, quenched with a saturated aqueous solution of NaHCO3 and the organic layer dried over Na2SO 4 . Silica gel column chromatography (hexane-EtOAc, 10:1) afforded 28a (1.735 g) and 280 (115 mg) in a total yield of 97% 30 (15:1 a/S ratio). Data for 28a. Rf (hexane-EtOAc, 5:1) = 0.40. [a]D + 126.1 (c 1.21, CHCl3). 1H NMR (200 MHz, CDCl,) d: 1.17 (s, 9H, 'Bu), 2.02 (s, 3H, CH 3 CO), 3.29 (m, 1H, H2), 3.97 - 4.07 (m, 1H, H 4 ), 4.21 - 4.37 (m, 5H, H1s,

H

6 a, H 6 b, CH 2 Ph), 4.37 (dd, J 3

,

4 = 8.8 Hz, J 3 ,2 = 2.8 Hz, 1H, WO 00/39141 PCT/GB99/04400 47 H3), 4.59 - 4.68 (bm, lH, CH 2 Ph), 4.97 - 5.18 (bm, 1H,

CH

2 Ph), 5.26 (d, J 1

,

2 = 1.5 Hz, 1H, H 1 ), 7.21 - 7.84 (m, 25H, ArH). "C NMR (50 MHz, CDCl 3 ) d: 19.32, 20.79, 27.17, 29.67, 63.57, 71.03, 71.83, 74.04, 74.08, 75.25, 76.13, 5 77.19, 79.59, 85.23, 127.28, 127.36, 127.42, 127.27, 127.76, 127.89, 127.97, 128.17, 128.26, 128.36, 128.81, 129.82, 129.96, 131.62, 133.11, 134.31, 136.08, 138.00, 138.26, 170.72. Anal. Calcd. for C 44

H

48

C

6 SS1: C, 72.10; H, 6.60; S, 4.37. Found: C, 72.31; H, 6.35; S, 4.12. 10 Phenyl 2,4-di-O-benzyl-3-O-(tert-butyldiphenylsilyl)-1 thio-a-D-mannopyranoside (29). To a solution of 100 mg (0.14 mmol) of 28a in 2 mL of methanol was added 0.4 mL of sodium methoxide in methanol (1M). The reaction 15 mixture was stirred for 1 h at room temperature, neutralized with Amberlite IR-120 H*, filtered and evaporated. Silica-gel column chromatography (hexane EtOAc, 3:1) afforded 29 (95 mg, quantitative yield). Rf (hexane-EtOAc, 3:1) = 0.46. M.p. = 46-48'C. [a]D + 131.1 20 (c 1.17, CHC1 3 ). 'H NMR (200 MHz, CDCl 3 ) d: 1.16 (s, 9H, 'Bu), 3.32 (m, 1H, H 2 ), 3.77 - 3.81 (m, 2H), 4.05 - 4.08 (m, 2H, H 4 ), 4.32 (dd, 2H, CH 2 Ph), 4.38 (dd, J 3

,

4 = 8.8 Hz, 73,2 = 3.1 Hz, 1H, H 3 ), 4.62 - 4.77 (bm, 1H, CH 2 Ph), 4.97 5.10 (bm, 1H, CH 2 Ph), 5.20 (d, J 1

,

2 = 1.7 Hz, 1H, Hi), 7.21 25 - 7.84 (m, 25H, ArH). 13C NMR (50 MHz, CDCl 3 ) d: 19.31, 27.16, 62.21, 72.24, 73.34, 73.89, 75.16, 76.02, 127.39, 127.41, 127.63, 127.71, 127.88, 128.23, 128.33, 128.90, 129.77, 129.92, 131.66, 133.21, 134.46, 136.07, 138.27. Anal. Calcd. for C 42

H

46

O

5 SSi: C, 73.10; H, 6.71; S, 30 4.64. Found: C, 73.12; H, 6.43; S, 4.37. 3-O-Benzyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-B-D glucopyranosyl fluoride (30). To a solution of 100 mg (0.17 mmol) of phenyl 3 -0-benzyl-4,6-0-benzylidene-2- WO 00/39141 PCT/GB99/04400 48 deoxy-2-phthalimido-1-thio-b-D-glucopyranoside ("I in 1.7 mL of CH 2 Cl 2 at -15 C, 68 mL (0.52 mmol) of diethylaminosulphur trifluoride were added dropwise followed by 46 mg (0.26 mmol) of NBS. The reaction 5 mixture was stirred for 4 h 30 min, quenched with a saturated solution of NaHCO 3 in water/ice, extracted with

CH

2 Cl 2 and dried over Na 2

SO

4 . Silica-gel column chromatography (hexane-EtOAc, 4:1) afforded 30 in quantitative yield. Rf (toluene-EtOAc, 10:1) = 0.56. 10 M.p.: 173-175 0 C. [aID + 62.0 (c 0.99, CHCl 3 ). 1H NMR (200 MHz, CDCl 3 ) d: 3.60 - 3.78 (m, 1H), 3.84 - 3.96 (m, 2H), 4.24 - 4.52 (m, 3H), 4.65 (dd, 2H, CH 2 Ph), 5.64 (s, 1H,

H

7 ), 5.90 (dd, 1H, Ji= 53.4 Hz, J1, 2 = 7.6 Hz, H 1 ), 6.84 7.80 (m, 14H, ArH). 13C NMR (50 MHz, CDCl 3 ) d: 41.98, 15 55.57, 55.98, 65.72, 65.82, 68.36, 73.74, 73.93, 74.15, 82.36, 101.48, 102.93, 107.22, 123.49, 126.04, 127.52, 128.07$ 128.30, 129.12, 131.51, 134.04, 137.04, 137.62. Anal. Calcd. for C 28

H

2 4 FN0 6 : C, 68.70; H, 4.94; N, 2.86. Found: C, 68.48; H, 5.10; N, 2.85. 20 Phenyl 0-(3-0-benzyl-4,6-0-benzylidene-2-deoxy-2 phthalimido-5-D-glucopyranosyl)-(1-6)-2,4-di-O-benzyl-3 0-(tert-butyldiphenylsilyl)-1-thio-a-D-mannopyranoside (31). A mixture of 475 mg (0.688 mmol) of 29, 800 mg 25 (2.68 mmol) of zirconocene dichloride, 1.39 g (5.37 mmol) of silver triflate and powdered 4A molecular sieves in 14 mL of CH 2 C1 2 was stirred in the dark and at room temperature for 30 min. At this time, the reaction mixture was cooled to -40'C and 437 mg (1.3 mmol) of 30 30 in 6 mL of CH 2 C1 2 were added dropwise over 30 min. After stirring for lh 30 min, the mixture was quenched with a saturated aqueous solution of NaHCO 3 , diluted with CH 2 Cl 2 , washed with brine, dried over Na 2

SO

4 , concentrated and chromatographed (diethyl ether/cyclohexane, 1:2) to yield WO 00/39141 PCT/GB99/04400 49 31 (654 mg, 82%). Rf (hexane-EtOAc, 3:1) = 0.36. M.p. 82-85-C. [at]D + 89.6 (c 0.95, CHC1 3 ). 'H NMR (500 MHz, CDC1 3 ) d: 0.99 (s, 9H, 'Bu), 3.15 (bs, 1H, H 2 d), 3.61 (dt, J5d,4d = J5d,6d = 9- 8 Hz, J5d,6d' = 4. 9 Hz, 1H, H 5 d) , 3. 70 5 3.79 (m, 4H), 3.99 - 4.08 (m, 3H), 4.17 - 4.25 (m, 3H), 4.30 - 4.36 (m, 2H), 4.41 - 4.54 (m, 2H), 4.63 (dd, 2H,

CH

2 Ph), 5.13 (bs, 1H, Hc), 5.29 (d, Jld,2d = 8.3 Hz, 1H, Hd) , 5.56 (s, 1H, H 7 d), 7.18 - 7.66 (m, 39H, ArH). "C NMR (50 MHz, CDCl 3 ) d: 19.21, 27.09, 55.57, 66.12, 68.48, 10 68.70, 71.57, 72.17, 73.72, 74.03, 74.63, 75.91, 76.66, 76.81, 77.20, 77.42, 78.00, 78.15, 79.58, 82.97, 85.27, 99.00, 101.30, 123.21, 126.07, 126.94, 127.18, 127.31, 127.51, 127.60, 127.99, 128.19, 128.27, 128.80, 128.97, 129.63, 129.84, 131.13, 131.65, 133.17, 133.59, 134.35, 15 134.89, 136.03, 137.42, 138.00, 138.31. Anal. Calcd. for

C

7

OH

69 NOnSSi: C, 72.45; H, 5.99; N, 1.21; S, 2.76. Found: C, 72.21; H, 6.10; N, 1.29; S, 2.57. 0-(3-0-Benzyl-4,6-0-benzylidene-2-deoxy-2-phthalimido-$ 20 D-glucopyranosyl)-(1-6)-2,4-di-O-benzyl-3-0-(tert butyldiphenylsilyl)-a-D-mannopyranose (32). To a solution of 105 mg (0.09 mmol) of 31 in 1.8 mL of acetone in the dark at -15*C, 24 mg (0.14 mmol) of NBS were added. Ten minutes later, the reaction mixture was 25 quenched with a saturated aqueous solution of NaHCO3, diluted and extracted with EtOAc, washed with brine and dried. Silica-gel column chromatography (hexane-EtOAc, 3:1) afforded 31 (91 mg, 94%). Rf (hexane-EtOAc, 3:1) = 0.15. M.p. 74-76'C. 'H NMR (200 MHz, CDCl 3 ) d: 1.04 (s, 30 9H, t Bu), 2.60 (d, JOHlc= 2.5 Hz, 1H, OH), 3.03 (Yt, J 2 c,1c = J2c,3c = 2.8 Hz, 1H, H2c), 3.41 - 3.90 (m, 7H), 4.11 4.80 (m, 12H), 5.55 (d, Jid, 2 d = 8.3 Hz, 1H, Hld), 5.60 (s, 1H, H 7 d), 7.10 - 7.71 (m, 34H, ArH). 13C NMR (50 MHz, CDCl 3 ) d: 19.22, 27.18, 55.88, 66.13, 68.19, 68.85, 72.51, WO 00/39141 PCT/GB99/04400 50 72.79, 73.21, 74.01, 74.36, 74.57, 74.65, 75.79, 76.67, 77.20, 77.40, 78.19, 78.32, 83.26, 92.32, 98.86, 101.38, 123.24, 126.09, 127.34, 127.48, 127.62, 127.68, 127.80, 128.19, 128.01, 128.15, 128.26, 128.97, 129.56, 129.69, 5 129.83, 131.56, 131.63, 133.53, 133.76, 133.91, 134.37, 136.12, 137.44, 137.80, 137.98, 138.59. Anal. Calcd. for

C

64

H

65 NOSi: C, 71.96; H, 6.13; N, 1.31. Found: C, 71.71; H, 5.85; N, 1.37. 10 0-(3-0-Benzyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-B D-glucopyranosyl)-(1-6)-2,4-di-O-benzyl-3-0-(tert butyldiphenylsilyl)-a-D-mannopyranosy1 trichloracetimidate (33). To a solution of 53 mg (0.05 mmol) of 32 in 0.25 mL of CH2Cl2 at room temperature, were 15 added 50 mL (0.50 mmol) of trichloracetonitrile and 7 mg (0.05 mmol) of flame-dried potassium carbonate. After 4 h, thed reaction mixture was diluted with CH2C1, filtered through celite and evaporated. Silica-gel column chromatography (hexane-EtOAc, 3:1) afforded 33 (53 mg, 20 88%) as a 13:1 a/S mixture. Data for 33a: Rf (hexane EtOAc, 3:1) = 0.36. M.p. 76-78-C. [a]D + 34.2 (c 0.61, CHCl 3 ) . 1H NMR (200 MHz, CDCl 3 ) d: 1.02 (s, 9H, 'Bu), 3.21 (m, 1H, H2), 3.56 - 3.87 (m, 6H), 4.04 - 4.62 (m, 9H), 4.64 (dd, 2H, CH2Ph) , 5.28 (d, J1d,2d = 8.2 Hz, 1H, Hid), 25 5.60 (s, 1H, H7d), 5.74 (m, 1H, Hic), 7.10 - 7.65 (m, 34H, ArH), 8.09 (s, 1H, NH). 1 3 C NMR (50 MHz, CDCl 3 ) d: 19.23, 27.13, 55.69, 66.09, 68.84, 72.19, 72.96, 73.87, 74.02, 74.76, 75.54, 76.23, 83.04, 95.81, 99.22, 101.32, 123.19, 126.08, 127.23, 127.29, 127.52, 127.67, 127.97, 128.19, 30 128.25, 128.96, 129.59, 129.80, 131.72, 133.08, 133.51, 134.27, 136.02, 137.44, 137.85, 138.04, 138.33, 159.80, 167.54. Anal. Calcd. for C 66

H

65 Cl3N2OSi: C, 65.37; H, 5.40; N, 2.31. Found: C, 65.10; H, 5.10; N, 2.07.

WO 00/39141 PCT/GB99/04400 51 Phenyl 6-O-Acetyl-2-O-benzyl-3, 4-O-isopropylidene-1-thio @-D-galactopyranoside (36). To a solution of 193 mg (0.48 mmol) of phenyl 2-0-benzyl-3,4-0-isopropylidene-1 thio--D-galactopyranoside (35) 45I in 0.77 mL of pyridine 5 and DMAP (cat.) at 0*C, was added dropwise 0.11 mL (1.20 mmol) of acetic anhydride. After stirring for 5 min at 0*C and 90 min at room temperature, the reaction mixture was evaporated. Silica-gel column chromatography (hexane-EtOAc, 4:1) of the crude afforded 36 (213 mg, 10 quantitative yield). Rf (hexane-EtOAc, 3:1) = 0.35. [cY]D + 9.2 (c 1.10, CHCl 3 ). 'H NMR (300 MHz, CDCl3) d: 1.35 (s, 3H, 'Pr), 1.41 (s, 3H, 'Pr), 2.06 (s, 3H, Ac), 3.54 (dd, J 2

,

3 = 6.2 Hz, J 2 ,1 = 9.4 Hz, 1H, H 2 ), 3.94 (dt, J 5

,

4 2.1 Hz, J 5

,

6 = 6.0 Hz, 1H, H), 4.19 (dd, J 4

,

5 = 2.0 Hz, J 4 ,3 15 = 5.8 Hz, 1H, H4), 4.28 (t, J 3

,

4 = 3

,

2 = 6.0 Hz, 1H, H3), 4.34 (d, J 6

,

5 = 6.1 Hz, 2H, H 6 a, H 6 b), 4.63 (d, Ji, 2 = 9.4 Hz, 1H, Hi), 4.76 (dd, 2H, CH2Ph), 7.25 - 7.57 (m, 10H, ArH) . 1 3 C NMR (50 MHz, CD 6 ) d: 20.33, 26.29, 27.73, 63.92, 73.52, 73.89, 74.42, 78.81, 79.89, 86.38, 110.27, 127.52, 20 127.83, 128.28, 128.92, 129.62, 130.02, 130.24, 132.58, 134.82, 138.73, 169.87. Anal. Calcd. for C2 4 H2,0 6 S: C, 64.85; H, 6.35; S, 7.21. Found: C, 65.17; H, 6.08; S, 7.25. 25 6-O-Acetyl-2-O-benzyl-3, 4-O-isopropylidene-D galactopyranose (37). To a solution of 115 mg (0.259 mmol) of 36 in 5 mL of acetone at -15 0 C was added 60 mg (0.336 mmol) of NBS and 5 mL (0.284 mmol) of water. After stirring for 10 min, the reaction mixture was 30 quenched with a saturated aqueous solution of NaHCO 3 , diluted and extracted with EtOAc, washed with brine and dried over Na 2

SO

4 . Silica-gel column chromatography (hexane-EtOAc, 3:1) afforded 37 (86 mg, 94%). Rf (hexane-EtOAc, 2:1) = 0.17. M.p. 122-124 0 C. 'H NMR (200 WO 00/39141 PCT/GB99/04400 52 MHz, CDCl.) d: 1.34 (s, 3H, 'Pra+b), 1.43 (s, 3H, 'Pr), 1.46 (s, 3H, 'Prb), 2.09 (s, 3H, Aca), 2.10 (s, 3H, Acb), 3.32 (d, JoHi= 6.4 Hz, 1H, OH), 3.52 (t, J 2 ,1 = J2,3 = 5.5 Hz, 1H, H 2 b), 3.67 (dd, J 2 ,1 = 3.8 Hz, J 2

,

3 = 5.7 Hz, 1H, 5 H 2 3), 4.08 (ddd, J = 2.1 Hz, J = 4.8 Hz, J = 7.0 Hz, 1H, Hsb), (dt, J = 4.3 Hz, J = 1.6 Hz, 1H, Hsa), 4.21 - 4.39 (m, 4H, H 3 b, H 4 , H 6 a, HEb), 4.45 (t, J = 6.0 Hz, 1H, H 3 3), 4.75 (dd, 2H, CH 2 Ph), 4.85 (dd, JiOH= 5.4 Hz, Ji, 2 = 7.9 Hz, 1H, Hib), 5.22 (dd, J 1

,

2 = 3.8 Hz, J3,OH = 6.3 Hz, 1H, 10 H 18 ), 7.29 - 7.39 (m, 5H, ArH). 1 3 C NMR (50 MHz, C 6 D) d: 13.38, 20.90, 25.58, 25.75, 27.15, 27.24, 63.74, 63.89, 66.85, 70.14, 72.96, 73.17, 73.41, 74.07, 75.36, 78.46, 90.57, 95.66, 109.95, 110.33, 127.92, 128.03, 128.18, 128.46, 128.58, 137.41, 137.70, 170.84. Anal. Calcd. for 15 C, 6 H2 4 0 7 : C, 61.36; H, 6.86. Found: C, 61.14; H, 6.66. 6-O-Adetyl-2-O-benzyl-3,4-O-isopropylidene-D galactopyranosyl trichloracetimidate (38). To a solution of 81 mg (0.230 mmol) of 37 in 1.2 mL of CH2C12 at room 20 temperature, were added 230.5 mL (2.30 mmol) of trichloracetonitrile and 76 mg (0.552 mmol) of flame dried potassium carbonate. After 5 h 45 min the reaction mixture was diluted with CH 2 C1 2 , filtered through celite and evaporated. Silica-gel column chromatography 25 (hexane-EtOAc, 6:1) afforded 38a (29 mg) and 38b (76 mg) (92% total yield). Data for 38B: Rf (hexane-AcOEt, 3:1) = 0.17. 'H NMR (300 MHz, CDCl3) d: 1.35 (s, 3H, iPr), 1.42 (s, 3H, iPr), 2.08 (s, 3H, Ac), 3.70 (dd, J 2 ,3 = 6.3 Hz, J2,1 = 7.3 Hz, 1H, H-2), 4.12-4.17 (m, 1H, H-5), 4.23 (dd, 30 J4,5 = 2.2 Hz, J 4

,

3 = 5.9 Hz, 1H, H-4), 4.30-4.38 (m, 3H, H 3, H-6a, H-6b), 4.34 (dd, 2H, CH 2 Ph), S.76 (d, Ji,2 = 7.6 Hz, 1H, H-1), 7.28-7.40 (m, 5H, ArH), 8.66 (s, 1H, NH). Data for 38a: Rf (hexane-AcOEt, 3:1) = 0.37. 'H NMR (300 MHz, CDCl3) d: 1.35 (s, 3H, 'Pr), 1.41 (s, 3H, 'Pr), 2.05 WO 00/39141 PCT/GB99/04400 53 (s, 3H, Ac), 3.81 (dd., J 2

,

3 = 6.8 Hz, J 2

,

1 = 3.5 Hz, 1H,

H

2 ), 4.23 - 4.49 (m, 5H, H 3 , H 4 , H5, H 6 a, HIb), 4.76 (dd., 2H, CH 2 Ph), 6.43 (d, J1, 2 = 3.6 Hz, 1H, H 1 ), 7.28 - 7.37 (m, 5H, ArH), 8.64 (s, 1H, NH). 5 Phenyl 0-(6-0-acetyl-2-0-benzyl-3,4-0-isopropylidene-a-D galactopyranosyl)-(1-6)-2-0-benzyl-3,4-0-isopropylidene 1-thio-p-D-galactopyranoside (39). A mixture of 64 mg (0.129 mmol) of 380, 45 mg (0.112 mmol) of 35 and 10 activated powdered 4A molecular sieves in 2.1 mL of ethyl ether was stirred for 90 min at room temperature. At this time, 155 mL (0.017 mmol) of a solution of trimethylsilyl triflate in ethyl ether (0.108M) were added. The reaction mixture was stirred for 45 min, 15 quenched with triethyl amine, diluted with CH 2 C1 2 , filtered through celite and evaporated in vacuo. Silica gel column chromatography (hexane-EtOAc) afforded 39a (62 mg) and 395 (10 mg) in a total yield of 86%. Data for 39a. Rf (hexane-EtOAc, 2:1) = 0.42. M.p.: 45-47'C. [alD + 20 48.3 (c 0.88, CHCl 3 ). 'H NMR (300 MHz, CAD 6 , 30 *C) d: 1.24 (s, 3H, 'Pr), 1.26 (s, 3H, 'Pr), 1.35 (s, 3H, 'Pr), 1.41 (s, 3H, 'Pr), 1.77 (s, 3H, Ac), 3.50 - 3.59 (m, 2H,

H

5 e, H 6 e), 3.69 (dd, J 2 e,3e = 6.3 Hz, J2.e,1 = 9.5 Hz, 1H, H 2 e), 3.71 (dd, 72f,3f = 7.7 Hz, J 2 ff = 3.5 Hz, 1H, H 2 f), 3.76 25 (dd, J4e,5e = 1.9 Hz, Je, 3 e = 5.7 Hz, 1H, H 4 e), 3.85 (dd, J4, 5f= 2.6 Hz, J 4 f, 3 f = 5.5 Hz, 1H, H 4 f), 4.05 (t, J 3

.,

4 . =

J

3 e, 2 e = 6.0 Hz, 1H, H 3 e) , 4.19 (dd, J 6 e', 6 e = 9.5 Hz, J 6 e,',se 7.0 Hz, 1H, H 6 e,), 4.44 (ddd, J5f,4f = 2.6 Hz, J 5 f, 6 f = 8.0 Hz, J 5 f, 6 f = 4.1 Hz, 1H, H 5 f), 4.46 - 4.63 (m, 2H, H 6 f, H 6 f,), 30 4.56 (dd, J 3 f, = 5.5 Hz, J 3 f, 2 f = 7.7 Hz, 1H, H3), 4.68 (d, Jie, 2 e = 9.5 Hz, 1H, Hie), 4.75 (dd, 2H, CH 2 Ph), 4.84 (dd, 2H, CH 2 Ph), 4.96 (d, J,a 2 = 3.5 Hz, 1H, Hjf), 7.01 - 7.64 (m, 15H, ArH). 3 C NMR (50 MHz, CDCl3) d: 20.82, 26.29, 27.72, 27.96, 63.35, 65.56, 66.90, 72.43, 73.37, 73.73, WO 00/39141 PCT/GB99/04400 54 74.90, 75.85, 76.18, 78.00, 79.59, 84.74, 96.68, 109.15, 110.14, 126.58, 127.76, 127.89, 128.19, 128.25, 128.32, 128.76, 129.76, 134.57, 137.65, 138.06, 170.59. Anal. Calcd. for C 40

H

48 0 11 S: C, 65.20; H, 6.57; S, 4.35. Found: C, 5 65.05; H, 6.54; N, 4.14. Data for 39S. Rf (hexane-AcOEt, 2:1) = 0.33. 1 H NMR (300 MHz, C 6 D, 30 'C) d: 1.33 (s, 3H, 'Pr), 1.35 (s, 3H, 'Pr), 1.37 (s, 3H, 'Pr), 1.42 (s, 3H, 'Pr), 2.09 (s, 3H, Ac), 3.39 (dd, J 2 f, 3 = 6.4 Hz, J 2 ff = 7.8 Hz, 1H, H 2 f), 3.55 (dd, J 2 e, 3 e = 6.1 Hz, J 2 ee = 9.2 Hz, 10 1H, H 2 e), 3.88 (dt, J , 6 f = J 5 f, 6 f = 6.1 Hz, J5f, = 2.0 Hz, 1H, H 5 ), 3.94 - 4.21 (m, 4H), 4.10 (dd, J 4 f, 5 f = 2.0 Hz,

J

4 f, 3 f = 5.7 Hz, 1H, H 4 ), 4.14 (t, J 3 f, 4 f = J 3 f, 2 f = 6.0 Hz, 1H, H 3 f) , 4.28 (t, J 3 e, 4 e = J3e,2e = 5.9 Hz, 1H, H 3 e) , 4.33 (d, J6e,5e = J6e',5e = 6.1 Hz, 2H, H 6 e, H 6 e' , 4.42 (d, Jf= 2 f 7.8 15 Hz, 1H, Hjf), 4.72 (d, Le, 2 e = 9.2 Hz, 1H, Hie), 4.66 - 4.84 (m, 4H, 2 CH 2 Ph), 7.16 - 7.53 (m, 15H, ArH) .

13 C NMR (50 MHz, GADC1 3 ) d: 20.85, 26.30, 27.66, 63.47, 69.09, 70.70, 73.39, 73.44, 73.87, 75.81, 77.96, 78.72, 79.16, 79.42, 86.00, 103.10, 110.12, 110.21, 127.01, 127.46, 127.73, 20 128.19, 128.28, 128.84, 131.12, 137.83, 138.22, 170.70. 0-(6-0-Acetyl-2-0-benzyl-3,4-0-isopropylidene-a-D galactopyranosyl)-(1-6)-2-0-benzyl-3,4-0-isopropylidene D-galactopyranose (40). To a solution of 233 mg (0.316 25 mmol) of 39a in 6.5 mL of acetone at -15 0 C, were added 73 mg (0.411 mmol) of NBS and 6.3 mL (0.348 mmol) of water. After stirring for 5 min, the reaction was quenched with a saturated aqueous solution of sodium bicarbonate. The mixture was diluted and extracted with EtOAc and washed 30 with brine. Silica-gel column chromatography (hexane EtOAc, 3:1) afforded 40 (203 mg, quantitative yield). Rf (hexane- EtOAc, 2:1) = 0.12. 'H NMR (200 MHz, CDCl 3 ) d: 1.30 (s, 3H, 'Pr), 1.33 (s, 3H, 'Pr), 1.38 (s, 3H, 'Pr), 1.40 (s, 3H, IPr), 2.05 (s, 3H, Ac,), 2.06 (s, 3H, Acb), WO 00/39141 PCT/GB99/04400 55 2.74 (s, 1H, OH), 3.37 (t, J 2 e,3e = J2e,ie = 6.3 Hz, 1H, H2eb), 3.52 (dd, J 2

,

3 f = 7.7 Hz, Jf,If = 3.6 Hz, 1H, H 2 ), 3.63 (dd, J 2 e, 3 e = 5.7 Hz, J 2 e,ie = 3.7 Hz, 1H, H 2 ea), 3.68 3.92 (m, 2H), 4.00 - 4.46 (m, 8H), 4.66 - 4.84 (m, 6H), 5 4.82 (m, 1H, Hiea), 7.26 - 7.38 (m, 10H, ArH). 13C NMR (50 MHz, CDCla) d: 20.85, 21.03, 25.78, 25.89, 26.34, 27.31, 27.41, 28.02, 63.71, 63.87, 65.38, 65.56, 67.34, 67.60, 67.88, 71.40, 72.26, 72.35, 72.97, 73.07, 73.50, 73.68, 74.20, 75.41, 75.88, 75.97, 78.31, 78.58, 79.22, 79.31, 10 79.54, 90.54, 96.20, 97.08, 97.28, 109.37, 109.41, 109.57, 109.93, 127.72, 127.81, 127.86, 127.97, 128.01, 128.14, 128.33, 128.50, 137.59, 137.99, 138.16, 138.23, 170.01. 171.75. 15 0-(6-O-Acetyl-2-0-benzyl-3, 4 -O-isopropylidene-a-D galactopyranosyl)-(1-6)-2-0-benzyl-3, 4 -0-isopropylidene D-galactopyranosyl trichloracetimidate (41). To a solution of 185 mg (0.287 mmol) of 40 in 1.5 mL of CH 2 Cl 2 , were added 288 mL (2.870 mmol) of trichloroacetonitrile 20 and 80 mg (0.574 mmol) of flame-dried potassium carbonate. The reaction mixture was stirred for 2 hours, diluted with CH2C1 2 and filtered through celite. The solvent was evaporated at reduced pressure and the crude was purified by silica-gel column chromatography (hexane 25 EtOAc, 4:1), affording 41a (69 mg) and 413 (111 mg) (80% total yield). Data for 41a. Rf (hexane-EtOAc, 2:1) = 0.49. 'H NMR (200 MHz, CDCl3) d: 1.31 (s, 3H, 'Pr), 1.32 (s, 3H, 'Pr), 1.37 (s, 3H, 'Pr), 1.39 (s, 3H, 'Pr), 2.04 (s, 3H, Ac), 3.51 (dd, J2f,3f = 7.7 Hz, J 2 f,if = 3.4 Hz, 1H, 30 H 2 f), 3.72 (dd, J 6

,,

6 , = 10.5 Hz, JEe, 5 e = 5.2 Hz, 1H, H 6 e), 3.80 (dd, J 2 e, 3 e = 6.6 Hz, J 2 e,le = 3.6 Hz, 1H, H 2 e), 3.88 (dd, JEe',,e = 10.5 Hz, J 6 e', 5 ,e = 7.1 Hz, 1H, H 6 e) , 4.14 (dd, J = 2.5 Hz, J = 5.6 Hz, 1H), 4.17-4.50 (m, 5H, H 3 e, H 3 f,

H

5 e, H 5 f), 4.30 (d, J 5 f, 6 f = 8.4 Hz, 2H, H 6 f, H 6 f ), 4.65 - WO 00/39141 PCT/GB99/04400 56 4.85 (m, 4H, 2 CH 2 Ph), 4.72 (d, Jif,2f = 3.5 Hz, 1H, Hf), 6.38 (d, Jle, 2 e = 3.6 Hz, 1H, Hie), 7.25 - 7.38 (m, 10H, ArH), 8.57 (s, 1H, NH). Data for 41P. Rf (hexane-AcOEt, 2:1) = 0.27. [aC]D + 66.8 (c 0.92, CHC1 3 ). 1 H NMR (200 5 MHz, CDCl 3 ) d: 1.32 (s, 6H, 'Pr), 1.38 (s, 3H, 'Pr), 1.39 (s, 3H, 'Pr), 2.05 (s, 3H, Ac), 3.53 (dd, J 2 1,3, = 7.5 Hz, J2,, = 3.4 Hz, 1H, H 2 ,), 3.67 (dd, J 2

,

3 = 6.1 Hz, J 2

,

1 = 7.7 Hz, 1H, H2), 3.71 (dd, J6a,6b = 10.0 Hz, JEa,S = 5.6 Hz, 1H,

H

6 a) , 3. 94 (dd, J6b,63 = 10 .3 Hz, J6b,5 = 6. 8 Hz, 1H, HEb), 10 4.08 - 4.38 (m, 8H, H 3 , H 3 1, H 4 , H 4 ,, H 5 , H 5 ,, H 6 a,, H 6 1b), 4.74 (dd, 2H, CH 2 Ph), 4.83 (dd, 2H, CH 2 Ph), 4.85 (d, J 1

,

2 = 3.4 Hz, 1H, H 1 .), 5.72 (d, J 1 2 = 7.8 Hz, 1H, H 1 ), 7.26 7.41 (m, 10H, ArH), 8.63 (s, 1H, NH). Anal. Calcd. for

C

36

H

44 C1 3

NO

12 : C, 54.80; H, 5.62; N, 1.77. Found: C, 55.00; 15 H, 5.76; N, 1.81. 0- (3-d-Benzyl-4, 6-0-benzylidene-2-deoxy-2-phthalimido- B D-glucopyranosyl)-(1-6)-O-[2,4-di-O-benzyl-3-0-(tert butyldiphenylsilyl) -a-D-mannopyranosyl] - (1-4) -O- [6-0- (2 20 azido-3, 6-di-O-benzyl-2-deoxy-at-D-glucopyranosyl) ] 2,3:4,5-di-O-cyclohexylidene-1-0-menthoxycarbonyl-1D-myo inositol (42). A mixture of 281 mg (0.232 mmol) of 33, 147 mg (0.165 mmol) of 20 and powdered 4A molecular sieves in 3.3 mL of ethyl ether was stirred for 45 min at 25 room temperature. At this time, 275 mL (0.030 mmol) of a solution of trimethylsilyl triflate in ethyl ether (0.108M) were added dropwise. The reaction mixture was stirred for 15 min, quenched with triethyl amine, diluted with CH 2 C1 2 , filtered through celite, evaporated in vacuo 30 and chromatographed (toluene/EtOAc 20:1) to yield 42 (261 mg, 81%), 33 (44 mg, 18% referred to starting 33), and unreacted 20 (22 mg, 15 %)). Rf (hexane-EtOAc, 3:1) = 0.46. M.p. = 104-107 0 C. [aC]D + 40.6 (c 1.31, CHCl 3 ) . 'H NMR (500 MHz, CDCl 3 ) d: 0.69 (d, 3H, Mnt), 0.81 (d, 3H, WO 00/39141 PCT/GB99/04400 57

CH

3 Mnt), 0.83 (d, 3H, CH 3 Mnt), 0.90 (s, 9H, 'Bu), 0.91 1.01 (m, 2H, Mnt), 1.08 - 1.15 (m, 1H, Mnt), 1.16 - 1.23 (m, 1H, Mnt), 1.30 - 1.70 (m, 23H, cyclohex., 3 Mnt), 1.85 - 1.92 (m, 1H, Mnt), 2.01 - 2.06 (m, 1H, Mnt), 2.75 5 (bs, 1H, H 2 c), 3.13 (dd, J21, 3 b = 9.6 Hz, J2b,1b = 3.4 Hz, 1H,

H

2 b), 3.42 - 3.55 (m, 8H), 3.57 (dd, J 5 a, 4 a = 10.9 Hz, J 5 a, 6 a = 8.4 Hz, 1H, H' 5 ), 3.60 - 3.69 (m, 4H), 3.65 (t, J4d, 3d J4d, 5 d = 8.9 Hz, 1H, H 4 d), 3.79 - 3.87 (m, 3H), 3.95 (dd, J4,, = 10.9 Hz, J 4 a,33 = 7.3 Hz, 1H, H 4 a), 4.08 (dd, J 6 a,sa = 10 8.4 Hz, J6a,Ia = 2.4 Hz, 1H, H 6 a), 4.12 - 4.18 (m, 2H), 4.21 (dd, J2d,3d = 10.3 Hz, J 2d,1d = 8.3 Hz, 1H, H2d), 4.32 (dd, J 3d, 2d = 10.3 Hz, J 3d, 4d = 8.9 Hz, 1H, H 3 d), 4.36 (t, J 3 a, 4 a J3a, 2 a = 7.3 Hz, 1H, Ha), 4.39 - 4.52 (m, 6H), 4.53 (dd, J23,3 = 6.9 Hz, J2al 5 = 4.0 Hz, 1H, H 2 3), 4.67 (dd, 1H, 15 CH 2 Ph), 4.87 (bs, 1H), 4.96 (dd, La, 2a 4.0 Hz, J, 6 a = 2.4 Hz, 1H, Hl,), 5.13 (d, Jid,2d = 8.3 Hz, 1H, Hid), 5.25 (d, Jib, 2 b = 3. 4 Hz, 1H, Hib) , 5. 44 (s, 1H, H 7 d) , 6.76 - 7. ( i, 44H, ArH). 1 3 C NMR (50 MHz, CDC1 3 ) d: 16.08, 19.31, 20.78, 21.92, 23.17, 23.60, 23.73, 23.90, 24.77, 25.06, 25.93, 20 26.97, 31.43, 34.08, 34.52, 36.16, 36.30, 36.68, 40.60, 47.00, 55.57, 62.68, 66.10, 68.73, 69.43, 70.44, 71.23, 72.09, 73.10, 73.25, 73.95, 74.66, 76.50, 76.70, 78.84, 79.28, 80.00, 82.91, 96.22, 98.07, 98.90, 101.24, 112.19, 113.49, 123.12, 126.08, 126.61, 126.77, 127.29, 127.51, 25 127.72, 127.91, 127.97, 128.10, 128.20, 128.26, 128.95, 129.69, 129.81, 131.54, 133.46, 133.62, 134.48, 136.07, 137.46, 137.75, 138.05, 138.59, 154.16. Anal. Calcd. for

C

1 1 3

H

1 3 0

N

4 0 2 3 Si: C, 69.95; H, 6.75; N, 2.89. Found: C, 69.78; H, 6.85; N, 2.72. 30 0-(3-O-Benzyl-4,6-0-benzylidene-2-deoxy-2-phthalimido-B D-glucopyranosyl)-(1-6)-O-(2,4-di-O-benzyl-3-aY-D mannopyranosyl)-(1-4)-O-[6-0-(2-azido-3,6-di-O-benzyl-2 deoxy-a-D-glucopyranosyl)]-2,3:4,5-di-O-cyclohexylidene- WO 00/39141 PCT/GB99/04400 58 1-O-menthoxycarbonyl-1D-myo-inositol (43). 6 mL of a 0.92M solution of tetrabutylammonium fluoride in THF buffered with acetic acid, were added to 71 mg (0.036 mmol) of 42. The reaction mixture was stirred for 10 5 days at 50 0 C, then cooled and quenched with water, diluted and extracted with CH 2 C1 2 and dried over Na 2

SO

4 . Silica-gel column chromatography (hexane-EtOAc, 3:1) afforded 43 (55 mg, 88% yield). Rf (hexane-EtOAc, 3:1) = 0.23. M.p. 103-105 0 C. [aOID + 37.9 (c 0.60, CHCl). 1 H 10 NMR (300 MHz, CD 6 ) d: 0.69 - 0.71 (m, 1H, Mnt), 0.81 (d, 3H, CH 3 Mnt), 0.95 (d, 6H, CH 3 Mnt), 0.87-1.80 (m, 24H), 1.99 - 2.03 (m, 2H, Mnt), 2.13 (d, J 3 c,OH= 9.5 Hz, 1H, OH), 2.15 - 2.27 (m, 2H, Mnt), 3.17 (dd, J2b,3b = 10.4 Hz, J2b,1b = 3.5 Hz, 1H, H 2 b), 3.49 - 3.53 (m, 2H), 3.62 - 3.65 (m, 15 1H), 3.67 (dd, J 2 c,3c = 3.3 Hz, J 2 c,c = 1.5 Hz, 1H, H 2 c), 3.72 - 3.89 (m, 4H), 4.18 (dd, J3b,2b = 10.2 Hz, J3b, 4 b = 9.0 Hz, 1HI HJb), 3.96 - 4.48 (m, 13H), 4.58 (dd, J 6a,a = 2.9 Hz, 1H, H 6 a), 4.68 (dd, J2,3, = 6.7 Hz, J 2 aa = 4.1 Hz, 1H,

H

2 a), 4,.56 - 4.91 (m, 9H), 5.33 (s, 1H, H 7 d), 5.40 (d, Jic,2c 20 = 1.4 Hz, 1H, Hic), 5.44 (dd, Jla,2a = 3.9 Hz, La,,6 = 3.1 Hz, 1H, Hia), 5.50 (d, Jid,2d = 8.1 Hz, 1H, H,...), 5.69 (d, J1b,2b = 3.5 Hz, 1H, Hib), 6.78 - 6.83 (m, 2H, ArH), 6.85 6.90 (m, 2H, ArH), 7.04 - 7.40 (m, 26H, ArH), 7.47 - 7.41 (m, 2H, ArH), 7.66 - 7.69 (m, 2H, ArH). "C NMR (50 MHz, 25 C 6

D

6 ) d: 16.53, 20.86, 22.01, 23.57, 23.94, 24.10, 24.35, 25.14, 25.42, 26.48, 31.46,34.23, 35.06, 36.67, 36.88, 37.08, 40.89, 47.50, 56.35, 63.21, 66.42, 68.57, 68.83, 69.84, 71.65, 71.81, 71.98, 72.06, 73.57, 73.83, 74.15, 74.87, 75.30, 76.32, 76.72, 77.04, 77.25, 77.43, 77.96, 30 79.20, 79.29, 80.45, 83.35, 97.12, 98.79, 99.26, 101.43, 112.15, 113.48, 118.92, 123.26, 126.65, 127.70, 128.98, 129.48, 129.66, 129.92, 132.16, 133.46, 138.32, 138.43, 138.62, 138.75, 139.30, 139.37, 154.90, 167.91. Anal. Calcd. for C 97 Hu 1 2

N

4 0 2 3 : C, 68.46; H, 6.63; N, 3.29. Found: WO 00/39141 PCT/GB99/04400 59 C, 68.11; H, 6.55; N, 3.33. 0-(6-0-Acetyl-2-O-benzyl-3, 4 -O-isopropylidene-a-D galactopyranosyl)-(1-6)-O-(2-0-benzyl-3,4-O 5 isopropylidene-a-D-galactopyranosyl)-(1-3)-O-[O-(3-0 benzyl-4,6-0-benzylidene-2-deoxy-2-phthalimido-B-D glucopyranosyl)-(1-6)-]-O-(2,4-di-O-benzyl-a-D mannopyranosyl)-(1-4)-O-[6-0-(2-azido-3,6-di-O-benzyl-2 deoxy-a-D-glucopyranosyl)]-2,3:4,5-di-O-cyclohexylidene 10 1-0-menthoxycarbonyl-myo-inositol (44). A solution of 94 mg (0.119 mmol) of 41b, 45 mg (0.026 mmol) of 43 and activated powdered 4A molecular sieves in 0.6 mL of ethyl ether was stirred for 90 min at room temperature. At this moment, 37 mL (0.004 mmol) of a solution of 15 trimethylsilyl triflate in ethyl ether (0.108M) were added. The reaction mixture was stirred for 45 min, quenched with triethyl amine, diluted with CH 2 Cl 2 , filtered through celite and evaporated in vacuo. Silica gel column chromatography (2 x cyclohexane-Et20, 5:2) 20 afforded 44a (44 mg) and 44P (7 mg) in 83% total yield, 45 (2.3 mg, 5% referred to starting 43), 46 (35.5 mg, 38% referred to starting 413), and 47 (2.8 mg, 8% referred to starting 41@). 44a: Rf (hexane-EtOAc, 3:1) = 0.26. M.p.: 93-96-C. [ax]D + 54.1 (c 0.880, acetone) . 1H NMR (500 25 MHz, CD 6 , 70'C) d: 0.62 - 0.71 (m, 1H, Mnt), 0.76 (d, 3H, CH3Mnt), 0.88 (d, 3H, CH 3 Mnt), 0.89 (d, 3H, CH3Mnt), 1.20 (s, 3H, !Pr), 1.29 (s, 3H, 'Pr), 1.33 (s, 6H, 'Pr), 0.84 1.78 (m, 24H), 1.74 (s, 3H, Ac), 1.89 - 1.93 (m, 2H, Mnt), 2.12 - 2.18 (m, 2H, Mnt), 3.35 (dd, J 2 b, 3 b = 10.4 Hz, 30 J2b,lb = 3.7 Hz, 1H, H2b), 3.44 - 3.46 (m, 1H), 3.49 (t, J = 9.9 Hz, 1H, Hd), 3.58 - 3.61 (m, 2H, H 2 e, Hd), 3.64 (dd, J2,1f = 3.4 Hz, J 2 f,3f = 7.6 Hz, 1H, H 2 ) , 3.67 - 3.74 (m, 2H, Ha), 3.96 (t, J = 9.2 Hz, 1H), 3.99 - 4.01 (m, 2H, He), 4.06 - 4.20 (m, 9H, Hf, Hd, H2c, H), 4.23 (t, J = 9.6 WO 00/39141 PCT/GB99/04400 60 Hz, 1H, Hb), 4.30 - 4.69 (m, 24H), 4.71 (dt, 1H, Mnt), 4.78 - 5.00 (m, 4H, CH 2 Ph), 4.94 (d, JC,2t = 3.1 Hz, 1H, Hlf), 5.13 (d, J = 3.0 Hz, 1H, He), 5.3 (s, 1H, H 7 d), 5.33 (t, Ja, 2 a = 3.5 Hz, 1H, Hl 2 ), 5.42 (m, 1H, Hld) , 5.58 (d, 5 Jib,2b = 3.7 Hz, 1H, Hib), 5.62 (d, Jc, 2 c = 1.8 Hz, 1H, Hic), 6.76 - 7.57 (m, 44H, ArH). 13 C NMR (75 MHz, C 6 D, 50 0 C) d: 16.67, 20.52, 20.84, 21.99, 23.79, 24.14, 24.24, 24.38, 25.28, 25.50, 26.45, 26.65, 26.99, 28.19, 30.12, 31.56, 34.38, 35.14, 36.75, 37.03, 37.23, 41.01, 47.63, 56.47, 10 63.15, 64.06, 66.51, 66.56, 67.35, 67.64, 68.93, 70.14, 71.34, 71.79, 72.63, 72.79, 73.52, 73.66, 73.89, 74.01, 74.24, 74.74, 74.93, 75.61, 76.22, 76.91, 77.06, 77.23, 77.48, 78.02, 78.16, 78.81, 79.31, 80.97, 83.42, 97.56 (ClN3), 97.94 (ClMan, C1Gal'), 99.36 (ClNPht), 99.43 15 (C1Gal), 101.57 (Benzylidene), 108.94 ('Pr), 109.56 ('Pr), 112.16 (CHex), 113.42 (CHex), 123.36, 126.69, 127.03, 127.20i 129.28, 129.36, 132.33, 133.51, 138.75, 138.87, 138.94, 139.03, 139.50, 139.65, 154.88 (carbonate), 167,97 (N'Pht), 169.99 (Npht). 44$: Rf (hexane/EtOAc 3:1) 20 = 0.25. IID 2 0 + 38.1 (c 0. 69, CHC1) .

1 H NMR (C 6

D

6 , 500 MHz, 50'C): d 0.71 - 1.76 (m, 25H, cyclohex, 5H Mnt), 0.72 (d, 3H, CH 3 Mnt), 0.84 (d, 3H, CH 3 Mnt), 0.86 (d, 3H,

CH

3 Mnt), 1.23 (s, 3H, 'Pr), 1.27 (s, 3H, 'Pr), 1.37 (s, 3H, 'Pr), 1.48 (s, 3H, 'Pr), 1.74 (s, 3H, CH 3 CO), 1.93 25 1.98 (m, 2H, Mnt), 2.16 - 2.20 (m, 2H, Mnt), 3.08 (dd,

J

2 b, 3 b = 10.2 Hz, J2b,lb = 3.7 Hz, 1H, H 2 b), 3.34 - 3.38 (m, 1H), 3.42 - 3.65 (m, 5H, 2 Hd), 3.68 - 3.75 (m, 2H, Hf), 3.85 - 4.86 (m, 39H, H 1 ), 4.91 - 4.97 (m, 4H, Hlf), 5.29 (s, 1H, H 7 d), 5.43 - 5.46 (m, 3H, Hl 2 , Hic, Hid), 5.48 (d, 30 J2b,lb= 3.8 Hz, 1H, Hib), 6.72 - 7.61 (m, 44H, ArH) . 13C NMR (CD , 75 MHz, 50 0C): d 16.4, 20.6, 20.9, 22.0, 23.5, 24.2, 25.3, 25.5, 26.4, 26.6, 26.7, 27.2, 28.0, 28.3, 30.1, 31.4, 34.3, 35.0, 36.7, 37.0, 40.9, 47.5, 56.3, 63.7, 64.1, 66.4, 68.8, 70.0, 71.5, 71.9, 72.4, 72.9, 35 73.1, 73.3, 73.5, 73.8, 74.2, 74.8, 75.0, 75.3, 75.7, WO 00/39141 PCT/GB99/04400 61 76.9, 77.5, 77.7, 79.1, 80.0, 83.5, 97.4 (C-lb), 97.7 (C lf), 99.0 (C-lc), 100.3 (C-le), 101.0 (C-ld), 101.5 (C 7d), 109.5 ('Pr), 109.8 ('Pr), 112.2 (cyclohex), 113.4 (cyclohex), 123.3, 126.7, 127.5, 128.0, 128.4, 129.3, 5 132.2, 133.5, 138.4, 138.7, 139.1, 139.4, 154.8 (OCO2), 168.9 (NCO), 170.0 (CH3CO). 45: Rf: 0.49 (hexane/EtOAc 2:1) . 'H NMR (CDC1, 300 MHz) : d 0.00 (s, 9H, (CH3)3Si), 0.78 - 2.09 (m, 29H, cyclohex, Mnt), 0.75 (d, 3H, CH3Mnt), 0.81 (d, 3H, CH3Mnt), 0.85 (d, 3H, CH3Mnt), 3.39 - 3.41 10 (m, 2H, H 2 b, H2,), 3.50 - 4.00 (m, 14H), 4.10 - 4.85 (m, 18H), 4.99 (dd, Jla,2 = 2.7 Hz, Jia, 6 a = 3.9 Hz, 1H, Hia), 5.16 (d, Jic,2c = 2.0 Hz, 1H, Hic), 5.20 (d, Jid,2d = 8.0 Hz, 1H, Hid), 5.30 (d, Jib,2b = 3.2 Hz, 1H, Hib), 5.53 (s, 1H, Hgd), 6.82 - 7.52 (m, 34H, ArH). 46: Rf: 0.27 15 (hexane/EtOAc 2:1). 'H NMR (CDC1, 200 MHz): d 1.29 (s, 3H, 'Pr), 1.34 (s, 3H, 'Pr), 1.35 (s, 3H, 'Pr), 1.50 (s, 3H, 'Pr), 2.06 (s, 3H, CH3CO), 3.47 (dd, J ,3f = 8.0 Hz,

J

2 f,lf = 3.3 Hz, 1H, H 2 f), 3.56 (dd, J 6

,

6 , = 10.3 Hz, J 6

,

5 = 4.3 Hz, 1H, H), 3.81 (dd, J6',6 = 10.2 Hz, J6, 5 = 7.7 Hz, 1H, 20 H 6 '), 3.93 (dd, J = 4.6 Hz, J = 3.2 Hz, 1H, H 4 ), 4.13 4.48 (m, 7H), 4.52 - 4.58 (m, 1H), 4.66 (m, 2H, CH2Ph), 4.74 (m, 2H, CH 2 Ph), 4.78 (d, Jff 2 = 3.5 Hz, 1H, Hlf), 5.65 (dd, Je, 2 e = 4.6 Hz, Le,NH = 8.5 Hz, 1H, Hie), 7.29 7.41 (m, 10H, ArH), 8.57 (s, JNH,1e = 8.5 Hz, 1H, NH). 47: 25 Rf: 0.38 (hexane/EtOAc 2:1). 'H NMR (CDC13, 300 MHz): d 1.28 (s, 3H, 'Pr), 1.46 (s, 3H, 'Pr), 3.51 (dd, J 6

,

6 = 7.6 Hz, J 6 ,5= 5.4 Hz, 1H, H), 3.52 (s, 1H, H2), 4.01 (d, J 6

',

6 7.6 Hz, 1H, H6'), 4.17 (d, J3, 4 = 7.0 Hz, 1H, H3), 4.39 (Yt, J4,3 = 6.8 Hz, J 4

,

5 = 6.0 Hz, 1H, H 4 ), 4.44 (Yt, J 5

,

4 = 5.7 30 Hz, J5, 6 = 5.4 Hz, 1H, H 5 ), 4.59 (dd, 2H, CH 2 Ph), 5.36 (s, 1H, H1), 7.18 - 7.30 (m, 5H, ArH). 0-(6-O-Acetyl-2-0-benzyl-3,4-0-isopropylidene-a-D galactopyranosyl)-(1-6)-O-(2-0-benzyl-3,4-0- WO 00/39141 PCT/GB99/04400 62 isopropylidene-a-D-galactopyranosyl)-(1-3)-O-[O-(3-0 benzyl-4,6-0-benzylidene-2-deoxy-2-phtalimido-B-D glucopyranosyl)-(1-6)-]-O-(2,4-di-O-benzyl-a-D mannopyranosyl)-(1-4)-O-[6-0-(2-azido-3,6-di-O-benzyl-2 5 deoxy-a-D-glucopyranosyl)]-2,3:4,5-di-O-cyclohexylidene 1-0-acetyl-1-D-MyO-inositol (48). A solution of 44a (11.5 mg, 4.94 mmol) in 3:2 tetrahydrofurane-methanol (2.2 mL) was treated at room temperature with 2.1 M aqueous lithium hydroxide (0.35 mL, 0.741 mmol). After 10 31h the reaction mixture was diluted with dichloromethane and washed with water, the water phase washed with dichloromethane and the combined organic phases dried over sodium sulphate and twice co-evaporated with toluene. The residue was suspended in chloroform (0.25 15 mL) and triethylamine (0.7 mL, 4.94 mmol) added to the suspension. After cooling at 0 0 C acetic anhydride (0.12 mL, 1.13 mmol) and dimethylamino pyridine were added. The reaction mixture was kept at room temperature for six days with further additions of triethyl amine and acetic 20 anhydride as above every 24 h. The reaction mixture was diluted with dichloromethane, washed with water, the aqueous phase washed with dichloromethane and the combined organic phases washed with a saturated aqueous solution of sodium chloride and dried over sodium sulfate 25 and evaporated. The residue was chromatographically purified. The NMR spectrum of the reaction mixture revealed it to be a 5.5:1 mixture of 48 and an intermediate compound and the mixture was therefore solved in chloroform (0.25 mL) treated with triethyl 30 amine (0.7 mL, 4.94 mmol), cooled at 0 0 C, treated with acetic anhydride (0.12 mL, 1.23 mmol) and dimethylamino pyridine and warmed at 40 0 C. The reaction mixture was kept at this temperature for four days, cooled at room temperature, and worked up as above to give 8.4 mg (78%) WO 00/39141 PCT/GB99/04400 63 of 48. 'H NMR (500 MHz, CD 6 , 50"C) d 1.27 (s, 3H, 'Pr), 1,35 (s, 3H,'Pr), 1.41 (s, 3H, 'Pr), 1.44 (s, 3H, 'Pr), 1.78 (s, 3H, CH 3 Co), 1.79 (s, 3H, CH 3 Co), 1.30-1.86 (m, 20H, cyclohex), 3.43 (dd, J2b, 3b= 10.0 Hz, J2b, b=3.

7 Hz, 5 1H, Hb), 3.48-3.58 (m, 2H), 3.63-3.71 (m, 2H, Hsd), 3.65 (dd, J2e, e= 3.5 Hz, J2e, 3e= 7.6 Hz, 1H, H2e) , 3.72 (dd, J 2 f, 1f= 3.6 Hz, J 2 f, 3f= 7.7 Hz, 1H, H 2 ), 3.77 (dd, J= 8.7 Hz, J= 10.2 Hz, 1H, H 5 a), 3.79 (dd, J= 6.0 Hz, J= 10.1 Hz, 1H), 4.04 (t, J= 8.9 Hz, 1H), 4.07-4.12 (m, 3H, H 4 e, H 4 d, 10 Hsd), 4.15 (dd, J= 2.4 Hz, J= 5.5 Hz, 1H, H 4 ), 4.17-4.21 (m, 2H), 4.22 (s, 1H, H 2 c), 4.25-4.36 (m, 7H, H 3 a, H 4 1,

H

3 b) , 4.38-4.49 (m, 6H, H 6 a, Had, H 3 e), 4.52-4.78 (m, 15H,

H

2 3, H 3 , H2d, H6d) , 4 .87 (d, 1H, CH 2 Ph) , 4.98 (dd, 2H, CH2Ph),. 5. 00 (d, Jlf, 2f= 3. 5 Hz, 1H, Hif),f 5. 13 (d, 1H, 15 CH 2 Ph), 5.20 (d, Jle, 2e= 3.4 Hz, 1H, Hi 8 ), 5.36 (s, 1H, H 7 d), 5.47-5.50 (m, 2H, H 18 , Hid), 5.60 (d, Jlb, 2b= 3.7 Hz, 1H, Hlb), 5.76 (d, J 1 ,, 2c= 2.0 Hz, 1H, Hi,), 6.80-7.66 (m, 44H, ArH). 13 C NMR (CA[,, 125 MHz, 50 0 C): d 20.2, 20.3, 23.6, 23.9, 20 24.0, 25.0, 25.1, 26.2, 26.8, 27.9, 28.0, 34.7, 36.7, 36.9, 37.0, 56.2, 62.7, 63.8, 66.2, 67.0, 67.3, 68.6, 71.4, 72.3, 72.4, 72.5, 73.1, 73.4, 73.6, 73.7, 74.0, 74.6, 75.3, 76.0, 76.6, 76.7, 76.8, 76.9, 77.2, 77.4, 78.0, 78.5, 80.6, 83.1, 97.4 (C1), 97.5 (C 1 ), 97.6 (C 1 ), 25 99.0 (C 1 ), 101.3 (Cld), 108.7 (Cipso), 110.0 (Cipso), 111.5 (Cipso), 113.0 (Cipso), 123.1, 126.4, 127.0, 127.1, 127.1, 127.2, 127.3, 127.3, 127.4, 127.5, 127.6, 127.6, 127.7, 127.8, 127.8, 127.9, 128.0, 128.0, 128.1, 128.2, 128.2, 128.2, 128.3, 128.4, 128.4, 128.7, 132.0, 133.3, 30 138.2, 138.5, 138.6, 138.6, 138.7, 139.2, 139.4, 169.1, 169.7. 0-(6-0-Acetyl)-2-0-benzyl-3,4-0-isopropylidene-a-D galactopyranosyl)-(1-6)-O-(2-0-benzyl-3,4-0- WO 00/39141 PCT/GB99/04400 64 isopropylidene-a-D-galactopyranosyl)-(1-3)-O-[O-(2 acetamido-3-0-benzyl-4,6-0-benzylidene-2-deoxy-B-D glucopyranosyl-(1-6)-]-O-(2,4-di-O-benzyl-a-D manopyranosyl)-(1-4)-O-[6-0-(2-azido-3,6-di-O-benzyl-2 5 deoxy-a-D-glucopyranosyl) 1-2,3:4, 5 -di-O-cyclohexylidene-1 O-acetyl-1-D-myo-inositol (49). To a solution of 48 (8.4 mg, 3.84 mmol) n-butyl alcohol (0.77 mL) ethylenediamine (167 mL, 2.50 mmol) was added at room temperature. After 90 minutes the temperature was raised to 90 0 C and the 10 reaction mixture was kept at this temperature for 18h, then cooled, evaporated and the residue co-evaporated twice with toluene. The residue was solved in chloroform (0.25 mL), treated with triethyl amine (0.8 mL, 5.76 mmol) and the solution cooled to 0*C before adding acetic 15 anhydride (0.25 mL, 2.69 mmol) and a catalytic amount of dimethylamino pyridine. The reaction mixture was allowed to warn and kept at room temperature for 20h, then the solvent was evaporated and the residue purified by column chromatography (2:1 hexane-ethyl acetate) to give pure 49 20 (7.5 mg, 93%) . 'HRMN (CD 6 , 500 MHz, 50'C) : d 1.29 (s, 3H, 'Pr), 1.38 (s, 3H, 'Pr), 1.42 (s, 6H, 2iPr), 1.78 (s, 3H,

CH

3 CO), 1.81 (s, 3H, CH 3 CO), 1.25-1.84 (m, 23H, cyclohex,

CH

3 CO), 3.22-3.31 (m, 1H, H2d), 3.34-3.39 (m, 1H, H 2 b), 3.47-3.53 (m, 1H, Hsd), 3.57 (t, J 6 d, 5d=J6d, 6d= 10.0 Hz, 1H 25 H 6 d) , 3.61-3.66 (m, 1H, H 4 d), 3.73-3.77 (m, 3H, H 5 a, H 2 e,

H

2 f), 3.79-3.84 (m, 1H, H 6 e), 3.86-3.90 (m, 1H), 4.06 (d, 76b, 6b= 10.2 Hz, 1H 6 b), 4.15-4.19 (m, 3H, H 4 f, H 6 d, H 6 e), 4.21-4.44 (m, 12H, H 4 e, Heb, H 3 a, H 4 a, Hab, H 2 c, H6a, H 3 c, H 4 ) 4.50-4.87 (m, 19H, Hsb, H 3 e, H 3 a, H 2 a, H3d, H 4 f, H 5 e), 4.98 (d, 30 1H, CH 2 Ph), 5.03-5.07 (m, 4H, CH 2 Ph, Hid, Hif), 5.21 (d, JNH, 2d= 6.7 Hz, 1H, NH), 5.38 (d, Le, 2e= 3.3 Hz, 1H, Hie), 5.40 (s, 1H, H' 7 d), 5.44 (d, 1H, CH 2 Ph), 5.45 (t, Ja, 2a= Ja, ,.= 3.8 Hz, 1H, H 18 ), 5.55 (d, Jib, 2b= 3.2 Hz, 1H, Hib), 5.91 (s, 1H, Hic), 7.05-7.65 (m, 40H, ArH).

WO 00/39141 PCT/GB99/04400 65 "C NMR (CED 6 , 125 MHz, 50 C): d 20.2, 20.3, 23.6, 23.9, 24.0, 25.0, 25.1, 26.2, 26.6, 27.9, 28.0, 34.7, 36.7, 36.9, 37.1, 59.1, 63.0, 63.8, 65.9, 66.3, 67.2, 67.4, 68.9, 69.4, 71.3, 71.4, 72.5, 72.6, 73.6, 73.6, 73.7, 5 73.8, 73.9, 74.4, 74.5, 74.7, 76.2, 76.6, 76.7, 76.8, 77.0, 77.4, 78.1, 80.7, 83.1, 97.4, (Cib), 97.7 (C1l, Cf), 98.1 (Cic), 100.3 (Cie), 101.3 (C 7 d), 108.8 (Cipso, 'Pr), 109.3 (Cipso, 'Pr), 111.5 (Cipso, cyclohex), 113.0 (Cipso, cyclohex), 126.4, 127.3, 127.4, 127.6, 127.8, 127.8, 10 127.9, 127.9, 128.0, 128.0, 128.1, 128.2, 128.2, 128.2, 128.2, 128.3, 128.3, 128.4, 128.4, 128.6, 138.3, 138.5, 138.6, 138.7, 139.2, 139.2, 169.1, 169.8. 0-(2-0-Benzyl-3, 4 -O-isopropylidene-a-D-galactopyranosyl) 15 (1-6)-O-(2-0-benzyl-3, 4 -O-isopropylidene-a-D galactopyranosyl)-(1-3)-O-[O-(2-acetamido-3-O-benzyl-4,6 O-benzylidene-2-deoxy-B-D-glucopyranosyl)-(1-6)--0-(2,4 di-O-benzyl-a-D-mannopyranosyl)-(1-4)-O-[6-0-(2-azido 3,6-di-O-benzyl-2-deoxy-a-D-glucopyranosyl)1-2,3:4,5-di 20 O-cyclohexylidene-1D-myo-inositol (50). To a solution of 49 (9.8 mg, 4.67 mmol) in 3:7 tetrahydrofuran-methanol (0.5 mL) a 0.2 M solution of sodium methoxide in methanol (70 mL, 14.0 mmol) was added at room temperature. After 8h the reaction mixture was neutralised with Dowex 50WX 25 resin, filtered and evaporated. The residue was purified by column chromatography (3:2 hexane-ethyl acetate) to give 50 (8.6 mg, 91%). 1 H RMN (CAlD, 500 MHz, 50C): d 1.31 (s, 3H, 'Pr), 1.33 (s, 3H, 'Pr), 1.39 (s, 3H, 'Pr), 1.43 (s, 3H, 'Pr), 1.22-1.43 (m, 5H, cyclohex), 1.45-1.75 30 (m, 18H, cyclohex,

CH

3 CO), 1.05 (s, 1H, OH), 2.69 (d, JOH, 1.= 2.3 Hz, 1H, OH), 3.24-3.28 (m, 1H, H 2 d), 3.30-3.34 (m, 1H, H 2 b), 3.48-3.53 (m, 1H, Hsd), 3.57 (t, J6d,5d= J6d,6d= 10.0 Hz, 1H, HEd), 3.61-3.66 (m, 1H, H 4 d), 3.73 (dd, Ja, a= 3.6 Hz, J 2 f, 3f= 7.4 Hz, 1H, H 2 f), 3.75 (dd, J= 8.5 Hz, J= 10.1 WO 00/39141 PCT/GB99/04400 66 Hz, 1H, H 5 a), 3.82 (dd, J2e, 1e= 3.4 Hz, J 2 e, 3e= 7.4 Hz, 1H,

H

2 e) , 3.85-3. 91 (m, 2H, H 4 c) , 3. 94-4. 01 (m, 2H, H 6 e) , 4. 04 (d, J 6 b, 6b= 11.3 Hz, lH 6 b), 4.10-4.16 (m, 3H, H 4 f, Hia, H 6 e), 4.19-4.23 (m, 3H, H 6 d), 4.24 4.04 (dd, 76b, 5b= 2.9 Hz, J 6 b, 5 6b= 11.1 Hz, lH 6 b) , 4.29-4.56 (m, 14H, H 2 3, H 38 , H 4 a, H 6 a, H 3 c, Hc, Hib, H4b, Hib, H 3 f, H 4 e) 4 65-4. 86 (m, 9H, Hae, Had, Hse), 4.99 (d, 2H, CH 2 Ph) , 5.07 (d, Ja, 2f= 3.5 Hz, 1H, Hif) , 5.08 (d, 1H, CH 2 Ph), 5.17-5.19 (m, 2H, Hid, Hib), 5.23 (d, JIH, 2d 7.1 Hz, 1H, NH), 5.41 (d, 1H, CH 2 Ph), 5.41 (s, 1H, H7d), 10 5.42 (d, Jle, 2e= 3.2 Hz, 1H, Hie), 5.83 (s, 1H, Hic), 7.14 7.65 (m, 40H, ArH) . "C NMR (CD 6 , 125 MHz, 33 0 C) : d 20.7, 23.2, 23.5, 23.8, 23.9, 24.0, 24.1, 25.0, 25.1, 26.2, 26.4, 27.9, 28.0, 33.6, 36.6, 36.6, 36.9, 59.2, 62.5, 62.8, 65.8, 67.4, 15 67.9, 68.9, 69.2, 71.3, 71.4, 72.6, 72.8, 73.0, 73.6, 73.8, 74.1, 74.4, 75.7, 76.1, 76.4, 76.4, 76.7, 76.8, 77.2, ,77.3, 77.4, 78.5, 80.6, 83.3, 96.8, (C1), 98.2 (C 1 ), 98.5 (C 1 ), 100.0 (C 1 ), 101.3 (C 7 d), 108.9 (Cipso, 'Pr), 109.1, (Cipso, 'Pr), 111.1 (Cipso, cyclohex), 126.5, 20 127.3, 127.4, 127.7, 128.1, 128.2, 128.3, 128.4, 128.6, 128.7, 138.3, 138.4, 138.7, 138.8, 139.3, 169.8.

WO 00/39141 PCT/GB99/04400 67 References: The references cited herein are all incorporated by reference. [1] G. Romero and J. Lasner, Advan. Pharmacol., 24 5 (1993) 21-50 and references therein. [2] I. Varela-Nieto, Y. Le6n, and H.N. Caro, Comp. Biochem. Physiol., 115B (1996) 223-241. [3] P. Stralfors, Bioessays, 19 (1997) 327-335. [4] M.C. Field, Glycobioloqy, 7 (1997) 161-168. 10 [5] D.R. Jones and I. Varela-Nieto, Tnt. J. Biochem. Cell Biol., 30 (1998) 313-326. [6] J.M. Mato, K. Kelly, A. Abler, L. Jairett, B.E. Corkey, B.E. Cashel and D. Zopf, Biochem. Biophys. Res. Commun., 146 (1987) 764-770. 15 [7] J. Larner, L.C. Huang, C.F.W. Schwartz, A.S. Oswald, T.Y. Shen, M. Kinter, G. Tang and K. Zeller, Biochem. Biophys. Res. Commun., 151 (1988) 1416 1426. [8] L.C. Huang, M.C. Fonteles, D.B. Houston, C. Zhang, 20 and J. Larner, Endocrinology, 132 (1993) 652-657. [9] J. Larner, P.J. Roach, L.C. Huang, G. Brocker, F. Murad and R. Hazen, Adv. Exp. Biol., 111 (1979) 103 112. [10] H.N. Caro, A Guadafno, M. Bernab6, M. Martin-Lomas, 25 J.M. Mato, R.A. Dwek and T.W. Rademacher, Glycoconiugate J., 10 (1993) 242. [11] G. Romero, G. G6mez, L. Huang, K. Lilley and L. Lutrell, Proc. Natl. Acad. Sci. USA, 87 (1990) 1476 1480. 30 [12] J. Represa, M.A. Avila, C. Miner, F. GisAldez, G. Romero, R. Clemente, J.M. Mato and I. Varela-Nieto, Proc. Natl. Acad. Sci. USA, 88 (1991) 8016-8019. [13] A. Zapata and M. Martin-Lomas, Carbohydr. Res., 234 (1992) 93-106. 35 [14] A. Zapata, Y. Le6n, J.M. Mato, I. Varela-Nieto,

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WO 00/39141 PCT/GB99/04400 68 Penart6s and M. Martin-Lomas, Carbohydr. Res., 264 (1994) 21-31. [15] C. Jaramillo, J.L. Chibra and M. Martin-Lomas, J. Org. Chem., 59 (1994) 3135-3141. 5 [161 N. Khiar and M. Martin-Lomas, J. Org. Chem., 60 (1995) 7017-7021. [17] H. Dietrich, J.F. Espinosa, J.L. Chiara, J. Jim6nez Barbero, Y. Le6n, I. Varela-Nieto, J.M. Mato, F.H. Cano, C. Foces-Foces and M. Martin-Lomas, Chem. Eur. 10 J., 5 (1999) 320-336. [18] R.D. Groneberg, T. Mizayaki, N.A. Stylianides, T.J. Schulze, W. Stahl, E.P. Schreiner, T. Suzuki, Y. Iwabuchi, A.L. Smith and K.C. Nicolau, J. Am. Chem. Soc., 115 (1993) 7593-7611. 15 [19] K.C. Nicolau, R.E. Dolle, D.P. Papakatjis and J.L. Randall, J. Am. Chem. Soc., 106 (1984) 4189-4192. [20] V, Pozsgay and H.J. Jennings, J. Org. Chem., 53 (1988) 4042-4052. [21] D.S.' Brocon, S.V. Ley, S. Vile and M. Thompson, 20 Tetrahedron, 47 (1991) 1329-1342. [22] D. Khane, S. Walker, Y. Cheng and van Engen, J. Am. Chem. Soc., 111 (1989) 6881-6882. [23] R.R. Schmidt and W. Kinzy, Advan. Carbohydr. Chem. Biochem., 50 (1994) 21-123. 25 [24] For a review, see B.V.L. Potter and D. Lampe, Angew. Chem. Int. Edn. Engl., 34 (1995) 1933-1979. [251 A. Zapata, R. Ferndndez de la Prahilla, M. Martin Lomas and S. Penart6s, J. Org. Chem., 56 (1991) 444 447. 30 [26] A. Aguil6, M. Martin-Lomas and S. Penart6s, Tetrahedron Lett., 33 (1992) 401-404. [27] S. David and S. Hanessian, Tetrahedron, 41 (1985) 643-663. [28] R.C. Mehrotra and V.D. Gupta, J. Organometal. Chem., 35 4 (1965) 2370- WO 00/39141 PCT/GB99/04400 69 [291 R. Kaster, K.L. Amen and W.V. Dahlhoff, Liebigs. Ann. Chem., (1975) 752. [30] K.M. Taba, R. Kbster and W.V. Dahlhoff, Synthesis, (1984) 399-401. 5 [31] P.J. Garegg, T. Ivessen, R. Johansson and B. Lindberg, Carbohydr. Res., 130 (1984) 322-326. [32] B. Kratzer, T.G. Meyer and R.R. Schmidt, Tetrahedron Lett., 34 (1993) 6881-6884. [33] R.H. Lemieux and R.M. Ratcliffe, Can. J. Chem., 57 10 (1979) 1244-1251. [34] N.V. Bovin, S.E. Zurabyan and A.Y. Khorlin, Carbohydr. Res., 98 (1981) 25 [35] H. Paulsen and W. Stenzel, Chem. Ber., 111 (1978) 2334 15 [36] M. Kloorterman, M.P. de Niijs and H. van Boom, J. Carbohydr. Chem., 5 (1986) 215 [37] A. Vasella, C. Witzig, J.L. Chiara and M. Martin Lomas, Hely. Chim. Acta, 74 (1991) 2073-2077. [38] R.J. Ferrier and R.H. Furneaux, Carbohydr. Res., 52 20 (1976) 63-68. [39] J. Gelas, Adv. Carbohydr. Chem. Biochem., 39 (1981) 71-156. [40] P.J. Garegg, H. Hultberg and S. Wallin, Carbohydr. Res., 108 (1982) 97-101. 25 [41] R. Johansson and B. Samuelsson, J. Chem. Soc. Perlaz. Trans. 1, (1984) 2371-2374. [42] M. Naruto, K. Ohno, N. Narusa and H. Takeuchi, Tetrahedron. Lett., (1979) 251-254. [43] E.J. Corey, H. Cho, C. RQcker and D.H. Hua, 30 Tetrahedron Lett., 22 (1981) 3455-3458. [44] E.J. Corey and A. Venkateswarlu, J. Am. Chem. Soc., 94 (1972) 6190-6191. [45] A. Fern~ndez-Mayoralas, A. Marra, M. Trumtel, A. Veysieres and P. Sinaij, Carbohydr. Res., 188 (1989) 35 81-95.

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Claims (28)

1. A compound represented by the general formula I: RO 5 RO d RO OOR~ YN RO eO OR RO eO O RO e RO 0 XN RO OR RO 10 R 2 f 0 R O-0--- OR RO f 0 RO OR 6 f wherein: 15 - each R is independently selected from hydrogen, an alkyl or substituted alkyl group, an acyl or substituted acyl group, a phosphate group or a protecting group, or two of the R groups may be cyclic phosphate; - NX group represents N 3 ; or in the NX group, X 20 represents one or more groups independently selected from hydrogen, alkyl or substituted alkyl, acyl or substituted acyl; - NY represents a phthalimido group (NPht) or N 3 ; or in the NY group, Y represents one or more groups 25 independently selected from hydrogen, alkyl or substituted alkyl, acyl or substituted acyl; with the proviso that the molecule contains no more than three phosphate groups; or a salt or derivative of said compound. 30

2. The compound of claim 1, wherein Rl" and/or R" are phosphate.

3. The compound of claim 1, wherein Rl" and R 2 1 together 35 are a cyclic phosphate group. WO 00/39141 PCT/GB99/04400 72

4. The compound of any one of claims 1 to 3, wherein NX is an NH 3 ' group.

5. The compound of any one of the preceding claims, 5 wherein Y is an AcH group.

6. The compound of any one of the preceding claims, wherein the R groups in one or more of units a, b, c and/or d are hydrogen or phosphate. 10

7. The compound of any one of the preceding claims, wherein the derivative is a coordination complex of the compound and a metal ion. 15

8. The compound of any one of the preceding claims, wherein the derivative is a prodrug.

9. The compound of claim 8, wherein the prodrug is a glycolipid derivative in which Ria is: 20 0 -P - - diacyl glycerol, wherein the prodrug is convertible to I following phospholipase cleavage. 25

10. The compound of any one of claims 1 to 6, wherein the compound is linked to a coupling partner.

11. The compound of claim 10, wherein the coupling 30 partner is a label, a supporting substrate, a carrier, an effector or inhibitor molecule or an immobilizer.

12. A composition comprising one or more of the compounds of any one of claims 1 to 11. 35 WO 00/39141 PCT/GB99/04400 73

13. A method of preparing a pharmaceutical composition, the method comprising admixing one or more of the compounds of any one of claims 1 to 11 with one or more pharmaceutically acceptable adjuvants and/or with one or 5 more other therapeutically active agents. 13. A compound of any one of claims 1 to 11 for use in a method of medical treatment. 10

14. Use of a compound of any one of claims 1 to 11 for the preparation of a medicament for the treatment of diabetes.

15. The use of claim 14, wherein the diabetes is obese 15 type II diabetes (NIDDM), diabetes due to insulin resistance, insulin resistance in type I diabetes and brittle diabetes and conditions associated with insulin resistance or insulin underproduction such as polycystic ovary disease. 20

16. Use of a compound of any one of claims 1 to 11 for the preparation of a medicament for the treatment of damage to the nervous system, motor neuron disease, neurodegenerative disorders or neuropathy. 25

17. The use of claim 16, wherein the damage to the nervous system is the result of one or more of trauma, stroke, surgery, infection, ischemia, metabolic disease, or toxic agents. 30

18. The use of claim 16, wherein the motor neuron disease is spinal muscular atrophy, paralysis or amyotrophic lateral sclerosis. 35

19. The use of claim 16, wherein the neurodegenerative WO 00/39141 PCT/GB99/04400 74 disorder is Parkinson's disease, Alzheimer's disease, epilepsy, multiple sclerosis, Huntingdon's chorea or Meniere' s disease.

20. A compound of the general formulae II, III, IV, or XI: RO OR 6 OR R O 1 4 bo 0j RO XN RO R ORV RO N OO RO d 0OR 2 C o 0 R a OR YNR O L IIIII IV R *2*0 R O YN OR R.O -O RO OR 0 0 x RO OR ~2~OR in which R, X and Y are as defined in claim 1 and each L is a leaving group which activates the anomeric position of the relevant saccharide unit in preparation for a glycosylation reaction with a glycosyl acceptor, or a leaving group precursor; or a salt or other derivative of said compound.

21. The compound of claim 20 represented by the general formula II, or a salt or other derivative thereof, in which Rlb (which is preferably hydrogen, or a protecting group chosen to permit orthogonal deprotection with respect to the other protecting groups present) is different to R 3 b and preferably also to R 6 b, and more WO 00/39141 PCT/GB99/04400 75 preferably Rb and R 6 b are the same as each other but different to R*.

22. A method of synthesis of a compound represented by 5 formula I, the method comprising: (a) preparing at least two of the intermediate disaccharide compounds II, III and IV; (b) condensing together the at least two disaccharides compounds II, III or IV to form a 10 tetrasaccharide intermediate compound; and, (c) reacting the tetrasaccharide intermediate with a third disaccharide intermediate selected from compounds II, III and IV to produce a compound of formula I. 15

23. The method of claim 22, wherein the three disaccharide compounds II, III and IV are reacted togther to form a compound represented by formula I.

24. The method of claim 22 or claim 23, further 20 comprising removing one or more of the R protecting groups after formation of the hexasaccharide.

25. The method of claim 22 which comprises reacting disaccharides II and II to form a tetrasaccharide 25 intermediate of the formula XI and further reacting the tetrasaccharide intermediate with a compound of formula IV.

26. A method of synthesis of a compound of formula II, 30 which involves: preparing a glycosyl donor, in the form of a 2 azido-2-deoxy-D-glucopyranosyl of formula VI (with X=N 2 ), from a 2-amino-2-deoxy D-glucosamine salt via a diazo transfer reaction from trifluoromethanesulphonyl azide 35 and then; WO 00/39141 PCT/GB99/04400 76 reacting that donor with a glycosyl acceptor in the form of a myo-inositol building block of formula V.

27. A method of synthesis of a tetrasaccharide of the 5 general formula XI, by reacting together a glycosyl acceptor of formula II, in which R 4 b is hydrogen, and a glycosyl donor of formula III, in which L is a leaving group. 10

28. A compound selected from compounds 9, 10, 11, 12, 13, 18, 19, 20, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 48, 49 or 50. 15