AU1060200A - Carbohydrates and methods for their synthesis - Google Patents

Description

WO 00/32615 PCT/GB99/03715 Carbohydrates and Methods for Their Synthesis Field of the Invention The present invention relates to carbohydrates and 5 methods for their synthesis, and more particularly to the synthesis of carbohydrates which are inositol phosphoglycan (IPG) mimetics using chiro-inositol as a starting material. S Background of the Invention Many of the actions of growth factors on cells are thought to be mediated by a family of inositol phosphoglycan (IPG) second messengers (Rademacher et al, 1994). It is thought that the source of IPGs is a "free" form of glycosyl phosphatidylinositol (GPI) situated in cell membranes. IPGs are thought to be released by the action of phosphatidylinositol-specific phospholipases following binding 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-like growth factor I (IGF-I), fibroblast growth factor, transforming growth factor B, 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, cell proliferation in the early developing ear and rat mammary gland. Soluble IPG fractions have been obtained from a variety of animal tissues including rat tissues (liver, kidney, muscle brain, adipose, heart) and bovine liver. IPG biological activity has also been detected in malaria parasitized RBC and mycobacteria. We have divided the family of IPG second messengers into distinct A and P type subfamilies on the basis of their biological WO00/32615 PCT/GB99/03715 2 activities. In the rat, release of the A- and P-type mediators has been shown to be tissue-specific (Kunjara et al, 1995). 5 WO98/11116 and WO98/11117 disclose the purification, isolation and characterisation of P and A-type IPGs from human tissue. Prior to these applications, it had not been possible to isolate single components from the tissue derived IPG fractions, much less in sufficient 0 quantities to allow structural characterisation. Accordingly, while some prior art studies describe the biological activities of the IPG containing fractions, speculation as to the identity of the active components from non-human sources of the fractions were based on 5 indirect evidence from metabolic labelling and cleavage techniques. A multistep synthesis of a P-type IPG mimetic from glucose has been previously reported in Jaramillo et al, ) 1994, which discloses a compound called C4, 1D-6-O-(2 amino- 2 -deoxy-a-D-glucopyranosyl)-chiro-inositol 1 phosphate. Tagliaferri et al, 1995, describes the production of lL 2,3:4,5-Bis-O-(tetraisopropyldisiloxane-1,3-diyl)-chiro inositol. However, the reaction to produce the intermediate has a very low yield (22%) and the reaction time is long (19 hours). The protected epoxide formed from the intermediate compound is further reacted to produce (-)-viburnitol, epoxides and aziridines. Summary of the Invention Broadly, the present invention relates to the synthesis of carbohydrates using chiro-inositol as a starting material, and especially to the synthesis of chiro- WO 00/32615 PCT/GB99/03715 3 inositol containing IPG mimetics such as compound C4. The compounds are the products of a glycosylation reaction between a glycosyl acceptor produced from chiro inositol and a glycosyl donor. 5 Accordingly, in a first aspect, the present invention provides a method for synthesizing a glycosyl acceptor from chiro-inositol, the method comprising: (a) protecting the trans-diequatorial hydroxyl 0 groups of chiro-inositol at positions 2, 3, 4 and 5; (b) reacting the intermediate from step (a) to produce an epoxide between hydroxyl groups at positions 1 and 6; and, (c) transdiaxially opening the epoxide to produce 5 the glycosyl acceptor. In a preferred embodiment, the method of producing a glycosyl acceptor from chiro-inositol comprises the steps of: D (a) protecting the trans-diequatorial hydroxyl groups of chiro-inositol at positions 2, 3, 4 and 5 to produce a trans-diaxial diol intermediate represented by the general formula; PO OH PO PC*) PO OH wherein P is a protecting group; (b) reacting the intermediate of step (a) to produce an epoxide between hydroxyl groups at positions 1 and 6 represented by the general formula; and, WO 00/32615 PCT/GB99/03715 4 P0 o PO P)\ PP PC0 (c) transdiaxially opening the epoxide with an alcohol having the formula R-OH to produce the glycosyl acceptor represented by the general formula; 5 PO OH

PC)

PO PO OR In some embodiments, the method comprises the further step of protecting the hydroxyl group at position 6 which is produced by the opening of the epoxide by the alcohol. D Preferably, the protecting groups P are bivalent, e.g. protecting the hydroxyl groups at positions 2 and 3, and 4 and 5. A preferred protecting group is tetraisopropyldisiloxane (TIPDS). In some embodiments, the method includes the further step of reacting the glycosyl acceptor to modify the identity of the protecting groups, e.g. to substitute TIPDS groups for a monovalent protecting groups such as Bn. This can improve the reactivity of the glycosyl acceptor to a glycosyl donor, e.g. to reduce the steric hindrance caused by a large protecting groups such as TIPDS. Further, the preferred method for the silylation of the 2,3,4,5 hydroxyl groups of chiro-inositol in dimethylformamide with TIPDS chloride (e.g. 2.5 equiv.) WO00/32615 PCT/GB99/03715 5 and catalytic 4 -dimethylaminopyridine (e.g. 0.4 equiv.) produced compound 2 in 76% yield in 2.5 hours. The use of naturally occurring D or L chiro-inositol as 5 the starting material for the synthesis of a glycosyl acceptor for use in glycosylation reactions, such as those used in the preparation of IPG-like compounds, has not been previously described. As the hydroxyl group to be glycosylated (6-OH) is axially oriented in the 0 preferred chair conformation of chiro-inositol, it is a challenging synthetic problem to achieve a good selectivity-reactivity balance in the glycosylation step. Further, the synthesis described herein employing chiro 5 inositol (e.g. D-chiro-inositol), instead of glucose as previously reported for the synthesis of C4 (Jaramillo et al, 1994), is advantageous as D-chiro-inositol is a readily available and relatively cheap starting material. This helps to avoid the time consuming multistep ) procedure of the prior art which synthesized this material from D-glucose. Further, the present invention reduces the number of steps required to produce the glycosyl acceptor to seven as compared to the eleven steps in the prior art procedure. The reactions are also S experimentally easier and less time consuming than those used in the prior art synthesis. In the synthesis of the glycosyl acceptor, an important step is the trans-diaxial opening of the epoxide, e.g. see the reaction of compound 3 to give compound 4 in scheme 1. This step is important as the synthesis is able to provide differentiation of positions 1 and 6 of chiro-inositol which due to the C2 symmetry of the molecule 1 are undistinguishable. The method achieves WO00/32615 PCT/GB99/03715 6 this selectivity in three steps making use of these symmetry properties (compound 4). The present invention further provides new intermediates 5 produced when the starting material is D-chiro-inositol. While, the steps leading to epoxide 3 (scheme 1) have been previously reported for the L-enantiomer (Taglafierri et al, 1995), in a further aspect, the present invention provides compounds having the general 0 formulae shown in steps (b) and (c) above, produced from D-chiro-inositol. In a further aspect, the present invention provides intermediates represented by the formulae 4, 5, 6, and 7. 5 The synthesis of the glycosyl acceptor shown in schemes 1 to 3 is equally applicable to using D-chiro-inositol or L-chiro-inositol as starting material. ) The glycosyl acceptor produced in scheme 1 can be reacted with any suitable glycosyl donor, e.g. mono, di-, tri-, tetra-, pentasaccharide units to form an oligosaccharide product, provided that the reactivity of that donor and the acceptor ensures that a balance is maintained between reactivity and selectivity. Thus, a glycan chain based on the chiro-inositol glycosyl acceptor can be enlarged with any suitable glycosyl donor, e.g. D-galactosamine or D-mannosamine could be used instead of D-glucosamine in scheme 3 resulting in different oligosaccharides in scheme 2. In a further aspect, the present invention provides a method of producing a chiro-inositol containing oligosaccharide comprising: WO00/32615 PCT/GB99/03715 7 reacting a glycosyl acceptor as defined above with a glycosyl donor to produce the chiro-inositol containing oligosaccharide. 5 Conveniently, this reaction can be carried out using a trichloroacetimidate derived glycosyl donor as described in the examples below. These reactions employ a glycosyl donor derivatised at position 1 which reacts with the hydroxyl group at position 6 of the glycosyl acceptor to 0 produce the chiro-inositol containing oligosaccharide. As indicated in scheme 2, the glycosylation reaction leads to a mixture of a and B glycosides, with the preferred a-configured glycosides as the major product. S Production of the B-glycoside could be preferred by varying reaction conditions, e.g. by using a different solvent or a different glycosyl donor other than an azido compound. ) Preferably, the protecting groups P used in step (a) is TIPDS chloride to favour the transdiaxial opening of the epoxide. By way of example, in the exemplified reaction scheme shown in scheme 1, 2 equivalents of TIPDS are used, with a first TIPDS molecule protecting the hydroxyls at positions 2 and 3 and a second TIPDS molecule protecting the hydroxyls at positions 4 and 5. Preferably, in step (c), the epoxide intermediate 3 is transdiaxially opened with an alcohol, more preferably an allyl alcohol as shown in scheme 1. Although the glycosylation reaction (scheme 2) may be carried out using intermediate 4 (scheme 1), preferably this intermediate is further reacted to reduce the steric hindrance of the protecting TIPDS groups, see for example WO00/32615 PCT/GB99/03715 8 the transformations 4 to 8 in scheme 1, to improve the reactivity of the glycosyl acceptor to the glycosyl donor, and hence improve the yield of the reaction. As shown in scheme 1, this results in the trans-diequatorial 5 hydroxyl groups at positions 2, 3, 4, and 5 being protected with Bn groups. The synthesis of the glycosyl donors 9 and 18 shown in scheme 3 can be carried out using a diazo transfer 0 reaction previously reported by Vasella et al, 1991. This procedure is much more convenient than the one used for compound 15 in Jaramillo et al, 1994, which is an alternative method of making the glycosyl donor. The standard manipulations leading from the products of the 5 diazo transfer reaction to several glycosyl donors have been indicated in the manuscript on A-type IPG mimetics. The use of a trichloroacetimidate as a glycosyl donor (scheme 2, compounds 9 and 18) is preferred. The pattern 3 of protective groups in these donors is important to achieve a good reactivity-selectivity balance. As shown in scheme 2 the stereoselectivity of the glycosylation does not seem to be greatly affected but the yield improves when acetyl groups are present as protecting groups in the glycosyl donor. Other alternative glycosylation reactions will be apparent to those skilled in the art. Preferably, the phosphorylation step (transformation 11 ) 12 in scheme 2) is performed using the phosphoramidite procedure. In the reaction between the glycosyl donor and acceptor, scheme 2 shows that the effect of using 9 and 18 is WO00/32615 PCT/GB99/03715 9 mainly on the yield of the reaction. However, compound 18 has the advantage that its preparation is simpler. In the reaction shown in scheme 2 (transformation 11-12), 5 the use of PdCl 2 /AcOH to remove the removal of the allyl group is advantageous as it accomplishes this reaction in a single step. Embodiments of the present invention will now be S described by way of example with reference to the accompanying reaction schemes. Brief Description of the Drawings Scheme 1 sets out the synthesis of glycosyl acceptor 8 from chiro-inositol. Scheme 2 sets out the reaction between glycosyl acceptor 8 and glycosyl donors 9 and 18. ) Scheme 3 sets out the synthesis of glycosyl donors 9 and 18. Detailed Description IPGs and IPG Analoques Studies have shown that 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 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 activity of insulin on adipocytes, whereas the P-type mediators mimic the glycogenic WO00/32615 PCT/GB99/03715 10 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 enhanced if the 5 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 0 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. 5 The ability of an anti-inositolglycan antibody to inhibit insulin action on human placental cytotrophoblasts and BC3H1 myocytes or bovine-derived IPG action on rat diaphragm and chick cochleovestibular ganglia suggests cross-species conservation of many structural features. ) However, it is important to note that although the prior 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. 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 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, also containing Mn 2 " and/or Zn 2 " ions and optionally WO00/32615 PCT/GB99/03715 11 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 5 fibroblasts in serum free medium, and stimulate pyruvate dehydrogenase phosphatase. Methods for obtaining A-type and P-type IPGs are set out in Caro et al, 1997, and in WO98/11116 and WO98/11117. 0 Pharmaceutical Compositions The mediators and analogues of the invention can be formulated in pharmaceutical compositions. These compositions may comprise, in addition to one or more of 5 the mediators, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials. well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier 0 or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. 5 Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal ) or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For intravenous, cutaneous or subcutaneous injection, or WO00/32615 PCT/GB99/03715 12 injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of 5 relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be 0 included, as required. Preferably, the pharmaceutically useful compound according to the present invention is given to an individual in a "prophylactically effective amount" or a 5 "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. Typically, this will be to cause a therapeutically useful amount of neurite growth or neuron proliferation, or the 3 prevention of a useful amount of neuron damage. The actual amount of the compounds administered, and rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. ) Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980. Compound C4 has been shown in WO99/38516 to be a P-type IPG mimetic and have the activity of stimulating neurite WO00/32615 PCT/GB99/03715 13 outgrowth in pure neuron and CVG cell cultures. The activity of this mimetic was shown to be as effective as P-type IPGs or insulin. 5 Abbreviations Bn: Benzyl TIPDS: Tetraisopropyldisiloxane MOM: Methoxymethyl Ac: Acetyl 0 General Methods All reactions were run under an atmosphere of dry argon using oven-dried glassware and freshly distilled and dried solvents. THF and diethyl ether were distilled 5 from sodium benzophenone ketyl. Dichloromethane and acetonitrile were distilled from calcium hydride. TLC was performed on Silica Gel GF 2 4 (Merck) with detection by charring with phosphomolibdic acid/EtOH. For flash chromatography, silica Gel (Merck 230-400 mesh) was used. D Columns were eluted with positive air pressure. Chromatographic eluents are given as volume to volume ratios (v/v). NMR spectra were recorded with a Bruker Avance DPX 3 0 0 (1H, 300 MHz), Bruker Avance DRX 400 ('H, 400 MHz), and Bruker Avance DRXs 5 00 (H, 500 MHz) spectrometers. Chemical shifts are reported in ppm, and coupling constants are reported in Hz. Routine spectra were referenced to the residual proton or carbon signals of the solvent. High-resolution mass spectra were recorded on a Kratos MS-80RFA 241-MC apparatus. Optical rotations ) were determined with a Perkin-Elmer 341 polarimeter. Elemental analyses were recorded on a leco CHNS-932 apparatus. The organic extracts were dried over anhydrous sodium sulfate and concentrated in vacuo. Compound 2 WO 00/32615 PCT/GB99/03715 14 Compound 1 (D-chiro-inositol, 1 g, 6.55 mmol, 1 equiv) was dissolved in dimethyl formamide (11 mL) with imidazol (1.5 g, 22 mmol, 4 equiv) and a catalytic amount of dimethylamino pyridine (274 mg, 2.22 mmol, 0.4 equiv) and 5 treated with TIPDSCl 2 (4.6 mL, 13.89 mmol, 2.5 equiv) dropwise. After 3.5 h the solution was diluted with ethyl acetate and washed successively with saturated ammonium chloride, water, and brine, dried over sodium sulfate, concentrated and the residue purified by column 0 chromatography (hexane-ethyl acetate 20:1) to give 2 (2.79 g, 76%) as a solid. 'H NMR (CDCl 3 , 500MHz): d 0.90-1.11 (m, 56H, iPr), 2.58 (s, 2H, OH), 3.83-3.87 (m, 2H), 3.95-3.98 (m, 2H), 4.03-4.06 (bd, 2H). 13C NMR (CDCl,, 125MHz): d 11.9, 12.0, 12.8, 12.9, 17.1, 17.2, 5 17.2, 17.3, 17.4, 17.4, 17.5, 71.8, 74.9, 76.5. Compound 3 Compound 2 (2.54 g, 3.89 mmol, 1 equiv) was dissolved in THF (64 mL) with triphenyl phosphine (5.07 g, 19.14 mmol, S 5 equiv) and treated at room temperature with DEAD (2.7 mL, 17.22 mmol, 4.5 equiv) dropwise. After 7 h the solvent was evaporated and the residue was purified by column chromatography [hexane, hexane-dichloro-methane (10:1- 4:1)] to give pure 3 (2 g, 80%) as a colorless glass. 1 H NMR (CDCl 3 , 500MHz): d 0.89-1.09 (m, 56H, 'Pr),3.14 (d, 1H, J=3.7 Hz), 3.33 (dd, 1H, J=1.9 Hz, J=2.8 Hz), 3.52 (dd, 1H, J=9.9 Hz, J=7.1 Hz), 3.61 (dd, 1H, J=7.7 Hz, J=9.8 Hz), 3.97 (d, 1H, J=7.1 Hz), 4.04 (dd, 1H, J=1.8 Hz, J=7.7 Hz) . 13C NMR (CDCl 3 , 125MHz): d 12.1, ) 12.7, 12.7, 12.8, 13.0, 17.0, 17.1, 17.1, 17.1, 17.2, 17.2, 17.3, 17.3, 17.3, 17.4, 17.5, 17.6, 56.2, 57.4, 73.4, 74.5, 75.4, 79.0.

WO 00/32615 PCT/GB99/03715 15 Compound 4 Compound 3 (2.2 g, 3.4 mmol, 1 equiv) was dissolved in dichloromethane (30 mL) with allyl alcohol (1.5 mL, 22.1 mmol, 6.5 equiv) then treated at room temperature with 5 boron trifluoride (1.5 mL, 11.9 mmol, 3.5 equiv). After 3 h the reaction was stopped with triethyl amine and the solvent evaporated and the residue purified by column chromatogrphy (hexane-ethyl acetate 70:1) to give pure 4 (1.84 g, 77%) as a syrup. 'H NMR (CDCl 3 , 500MHz): d 0 0.85-1.20 (m, 56H, iPr), 2.56 (s, 1H, OH), 3.76-3.80 (m, 2H), 3.90-3.93 (m, 3H), 3.99 (dd, 1H, J=2.9 Hz, J=9.2 Hz), 4.12 (dd, 1H, J=13.3 Hz, J=5.8 Hz, All), 4.34 (dd, 1H, J=4.9 Hz, J=13.3 Hz, All), 5.11 (dd, 1H, J=1.3 Hz, J=10.4 Hz, All), 5.21 (dd, 1H, J=1.6 Hz, J=17.2 Hz, All), 5 5.82-5.90 (m, 1H, All). Compound 5 MOMC1 (1.125 mL, 14.8 mmol, 6.25 equiv) in dioxane (3.5 mL) was treated with sodium iodide (1.78 g, 11.8 mmol, 5 0 equiv) and the mixture stirred for ten minutes at room temperature. Compound 4 (1.67 g, 2.36 mmol, 1 equiv) in dioxane (1.5 mL) and diisopropyl ethyl amine (2.76 mL, 16.34 mmol, 6.9 equiv) was added to the above solution and the mixture was heated at 85oC overnight. The 5 solution was cooled at room temperature, diluted with methylene chloride and washed with sodium bicarbonate, water, and brine, and concentrated. The residue was purified by column chromatography (hexane-ethyl acetate 50:1) to give pure 5 (1.43 g, 82%) as a syrup. H NMR ) (CDCl 3 , 500MHz): d 0.89-1.07 (m, 56H, iPr), 3.34 (s, 3H, MOM), 3.63 (t, 1H, J=3.6 Hz), 3.79-3.88 (m, 4H), 3.95 (dd, 1H, J=2.7 Hz, J=8.9 Hz), 4.08-4.12 (m, 1H, All), 4.31-4.36 (m, 1H, All), 4.74 (dd, 2H, MOM), 5.11 (dd, 1H, J=1.5 Hz, J=10.4 Hz, All), 5.20-5.24 (m, 1H, All), WO 00/32615 PCT/GB99/03715 16 5.82-5.90 (m, 1H, All). " 3 C NMR (CDCl 3 , 125MHz): d 12.0, 12.1, 12.2, 12.7, 12.7, 12.8, 12.8, 17.1, 17.3, 17.4, 17.4, 17.4, 17.4, 17.5, 55.3, 73.3, 74.4, 75.0, 76.9, 77.0, 79.0, 97.7, 115.9, 135.5. 5 Compound 6 To a solution of 5 (1.17 g, 1.56 mmol, 1 equiv) in THF (22 mL) at room temperature was added a solution IM of TBAF in THF (6.25 mL, 6.25 mmol, 4 equiv). The reaction 0 was stirred for 2h and then 40 mL of Pyr were added. The reaction mixtured was cooled to 0 0 C followed by the addition of 18 mL of acetic anhydride. After 18 h at room temperature the crude was poured on a mixture of water/ice and extracted with EtOAc. The organic layer 5 were washed with brine and dried over sodium sulfate. The residue was purified by column chromatography to obtain 675 mg of 6 (quantitative). 1 H NMR (CDCl 3 , 300MHz): d 1.99 (s, 6H, Ac), 2.03 (s, 3H, Ac), 2.04 (s, 3H, Ac), 3.39 (s, 3H, MOM), 3.91 (dd, 1H, J=3.1 Hz, J=4.3 0 Hz), 4.07-4.13 (m, 3H, 2 All), 4.62 (dd, 2H, MOM), 5.16-5.30 (m, 4H, 2All, H-5, H-2), 5.40-5.47 (m, 2H, H-3, H-4), 5.79-5.90 (m, 1H, All). 3 C NMR (CDCl 3 , 500MHz): d 20.6, 20.8, 20.9, 70.3, 70.4, 70.6, 71.2, 72.6, 73.7, 75.1, 97.4, , 117.9, 134.0, 169.1, 169.3, 169.4. 5 Compound 7 :- From compound 5: Compound 5 (318 mg, 0.425 mmol, 1 equiv) was disolved in THF (4.25 mL) then treated with TBAF (1 M, 0.85 mL, 0.85 3 mmol, 2 equiv). After 30 min, DMF (7 mL) was added followed by sodium hydride (136 mg, 3.4 mmol, 8 equiv) and benzyl bromide (303 mL, 2.55 mmol, 6 equiv). As no benzylation reaction took place, the mixture was filtered through silica gel and benzylated in the above conditions WO00/32615 PCT/GB99/03715 17 at 450C for 18 h. The mixture was diluted with ethyl acetate and washed with ammonium chloride, water, and brine and concentrated and the residue was purified by column chromatography (hexane-ethyl acetate 6:1) to give 5 pure 7 (102 mg, 38%). :- From compound 6: To a solution of 6 (650 mg, 1.50 mmol, 1 equiv) in 30 mL of MeOH at room temperature was added a solution 0.95M of 0LO NaOMe in MeOH (1.3 mL, 1.20 mmol, 0.8 equiv). After 30 min the solvent was evaporated and the crude coevaporated twice with toluene. The crude was solved in 21 mL of DMF and then NaH (481 mg, 12.03 mmol, 8 equiv) and benzyl bromide (1.07 mL, 9.03 mmol, 6.0 equiv) were added. The 5 reaction was stirred overnight and stopped with methanol, diluted with EtOAc, washed with water and brine and dried over sodium sulfate. The residue was purified by column chromatography (hexane-ethyl acetate 10:1) to give pure 7 (778 mg, 83%). 1 H NMR (CDCl 3 , 500MHz): d 3.18 (s, 3H, 0 MOM), 3.68-3.70 (m, 1H), 3.78-3.88 (m, 5H), 3.93-3.98 (m, 1H, All), 4.12-4.16 (mn, 1H, All), 4.54-4.91 (m, 10H,

CH

2 Ph, MOM), 5.11-5.13 (m, 1H, All), 5.16-5.20 (m, 1H, All), 5.78-5.83 (mn, 1H, All), 7.22-7.35 (m, 20H, ArH). 13C NMR (CDCl 3 , 125MHz): d 55.6, 72.2, 73.3, 73.4, 74.0, 75.7, 5 75.9, 79.3, 79.8, 82.1, 82.1, 97.3, 117.0, 127.4, 127.4, 127.6, 127.7, 127.9, 127.9, 128.0, 128.0, 128.1, 128.3, 128.3, 128.3, 128.4, 135.0, 138.5, 138.7, 139.1, 139.2. Compound 8 0 Compound 7 (730 mg, 1.17 mmol, 1.0 equiv) was dissolved in dichloromethane (11.7 mL) at -10 0 C then treated with thiophenol (0.156 mL, 1.52 mmol, 1.3 equiv) and boron trifluoride (0.148 mL, 1.17 mmol, 1.0 equiv). After 30 min the reaction was quenched with sodium bicarbonate and WO 00/32615 PCT/GB99/03715 18 extracted with methylene chloride. After drying over sodium sulfate and evaporation the residue was purified by column chromatography (toluene-ethyl acetate 14:1) to give pure 8 (550 mg, 81%). 1H NMR (CDC1 3 , 500MHz): d 2.40 5 (s, 1H, OH), 3.73-3.92 (m, 5H), 3.99-4.03 (m, 2H, H-6, All), 4.18-4.22 (m, 1H, All), 4.62-4.91 (m, 8H, CH 2 Ph), 5.13 (dd, 1H, J=1.4 Hz, J=10.4 Hz, All), 5.18-5.22 (m, 1H, All), 5.80-5.86 (m, 1H, All), 7.23-7.35 (m, 20H, ArH). 1 3 C NMR (CDCl 3 , 125MHz): d 68.2, 72.6, 73.3, 73.3, 0LO 75.6, 75.8, 75.9, 79.9, 80.3, 80.8, 81.6, 82.0, 116.9, 127.4, 127.5, 127.5, 127.7, 127.7, 127.7, 127.7, 127.9, 127.9, 127.9, 128.0, 128.3, 128.3, 128.3, 128.3, 128.4, 128.4, 128.5, 135.1, 138.2, 138.7, 139.1, 139.0, 139.1. 5 Compound O1A: A) Using 181 as glycosyl donor A mixture of compound 8 (197 mg, 0.339 mmol, 1 equiv), compound 18 ((490 mg, 1.04 mmol, 3 equiv) and 4 A molecular sieves in 3 mL of CH 2 Cl 2 was stirred at room 0 temperature for 45 min. After this time a solution 0.21M of TMSOTf en CH 2 Cl2 was added (160 mL, 0.034 mmol, 0.1 equiv). After lh the reaction was quenched with Et 3 N, filtrated through celite and evaporated to dryness. Column chromatography (cyclohexane-EtOAc 3:1) afforded 5 133 mg (44%) of 10At and 93 mg (31%) of 10AB. Data for 1A : 1H NMR (CDCl 3 , 500MHz): d 1.99 (s, 3H, Ac), 2.00 (s, 3H, Ac), 2.08 (s, 3H, Ac), 3.55 (dd, 1H, J=3.7 Hz, J=10.4 Hz, H-2'), 3.58 (dd, 1H, J=2.0 Hz, J=10.7 Hz, H-6'), 3.70-3.71 (m, 1H), 3.81-3.91 (m, 5H, H-6'), 3.97-4.01 (m, 0 2H, All), 4.20-4.24 (m, 1H, All), 4.33-4.37 (m, 1H, H-5'), 4.78 (d, 1H, J=3.6 Hz, H-1'), 4.59-4.94 (m, 8H,

CH

2 Ph), 4.98 (Yt, 1H, J=10.2 Hz, J=9.5 Hz, H-4'), 5.14-5.17 (m, 1H, All), 5.18-5.22 (m, 1H, All), 5.37 (Yt, 1H, J=9.4 Hz, J=10.2 Hz, H-3'), 5.78-5.85 (m, 1H, All), WO00/32615 PCT/GB99/03715 19 7.24-7.36 (m, 20H, ArH). " 3 C NMR (CDCl 3 , 125MHz): d 68.2, 72.6, 73.3, 73.3, 75.6, 75.8, 75.9, 79.9, 80.3, 80.8, 81.6, 82.0, 116.9, 127.4, 127.5, 127.5, 127.7, 127.7, 127.7, 127.7, 127.9, 127.9, 127.9, 128.0, 128.3, 128.3, 5 128.3, 128.3, 128.4, 128.4, 128.5, 135.1, 138.2, 138.7, 139.1, 139.0, 139.1. To a solution of 10Aa (140 mg, 0.156 mmol, 1 equiv) in methanol (3.1 mL) was added sodium methylate (0.95M, 10 0.165 mL, 1 equiv) at room temperature. The reaction mixture was stirred for 10 min and then neutralized with Amberlite resin IR-120H'. After filtration and evaporation of the solvent, the residue was dissolved in DMF (2.3 mL), and NaH (60% in oil, 39 mg, 0.97 mmol, 6 L5 equiv) and benzyl bromide (87 mL, 0.73 mmol, 4.5 equiv) were added. The reaction mixture was stirred for 1 h, then quenched with MeOH at 0 0 C, diluted with methylene chloride, washed with saturated aqueous sodium bicarbonate solution. The organic layer was dried over 0 sodium sulfate, evaporated under vacuum and the residue purified over column chromatography to give pure 10 (161 mg, 99%). B) Using 92 as glycosyl donor: 5 Compound 8 (73 mg, 0.125 mmol, 1.6 equiv) was mixed with compound 9 (49 mg, 0.078 mmol, 1 equiv) and coevaporated two times with toluene, then dissolved in methylene chloride (1 mL). Trimethylsilyl triflate (50 mL of a 0.1 M solution in methylene chloride) was added at -25 0 C. 0 After 30 minutes at -25 0 C, the mixture was allowed to warm during 10 minutes. The reaction was then stopped by addition of triethyl amine and evaporated to dryness. The residue was purified by column chromatography [hexane-ethyl acetate (7:1-3:1)] to give 35 mg (35%) of WO 00/32615 PCT/GB99/03715 20 10a and 25 mg (25%) of 103. Data for 10a: IH NMR (CDCl 3 , 500MHz): d 3.13 (dd, 1H, J=1.7 Hz, J=10.9 Hz, H-6'), 3.35 (dd, 1H, J=2.3 Hz, J=10.9 Hz, H-6'), 3.48 (dd, 1H, J=3.6 Hz, J=10.1 Hz, H-2'), 3.71 (t, 1H, J=9.5 Hz, H-4'), 5 3.74-3.88 (m, 5H, H-3'), 3.94-4.02 (m, 3H, All), 4.10-4.13 (m, 1H, H-5'), 4.18-4.21 (m, 1H, All), 4.24-4.92 (m, 14H, CH 2 Ph), 4.75 (d, 1H, J=3.6 Hz, H-I'), 5.14 (dd, 1H, J=1.2 Hz, J=10.4 Hz, All), 5.18 (dd, 1H, J=1.5 Hz, J=17.2 Hz, All), 5.77-5.85 (m, 1H, All), 0 7.05-7.48 (m, 35H, ArH). 13C NMR (CDCl 3 , 125MHz): d 63.8, 67.6, 70.7, 72.5, 73.1, 73.3, 73.5, 73.6, 74.2, 74.9, 75.5, 75.8, 76.1, 78.2, 78.3, 80.0, 80.5, 81.8, 82.0, 96.9, 117.3, 127.4, 127.4, 127.5, 127.7, 127.8, 127.9, 128.0, 128.0, 128.0, 128.1, 128.1, 128.1, 128.2, 128.3, 5 128.3, 128.3, 128.4, 128.4, 128.5, 134.9, 137.8, 137.9, 138.1, 138.6, 138.8, 139.0, 139.0. Compound 9 was prepared as indicated in scheme 2. This synthetic route is describe below. 0 Compound 11 A solution of sodium acetate (89.11 mg, 1.09 mmol, 8 equiv) in 20:1 acetic acid-water (3.4 mL) was degassed under Ar then added over 10a (141 mg, 0.135 mmol, 1 5 equiv) and palladium chloride (73.24 mg, 0.407 mmol, 3 equiv). The reaction was stirred for 6h 30 min then diluted with methylene chloride, filtered over Celite, and neutralized with sodium bicarbonate and washed with water and brine. After drying and evaporation the D residue was purified by column chromatography (hexane-ethyl acetate 5:1) to give pure 11 (100 mg, 74%). 'H NMR (CDCl 3 , 500MHz): d 2.46 (s, 1H, OH), 3.06 (dd, 1H, J=1.9 Hz, J=11.0 Hz, H-6'), 3.30 (dd, 1H, J=2.4 Hz, J=11.0 Hz, H-6'), 3.47 (dd, 1H, J=3.6 Hz, J=10.1 Hz, WO 00/32615 PCT/GB99/03715 21 H-2'), 3.71 (Yt, 1H, J=9.2 Hz, J=10.0 Hz, H-4'), 3.77 (Yt, 1H, J=8.7 Hz, J=9.3 Hz, H-3'), 3.80-3.87 (m, 3H, H-3, H-4, H-5), 3.94 (dd, 1H, J=9.1 Hz, J=3.2 Hz, H-2), 4.04 (t, 1H, J=3.6 Hz, H-1), 4.09-4.12 (m, 1H, H-5'), 5 4.16 (Yt, 1H, J=3.6 Hz, J=3.2 Hz, H-6), 4.22 (d, 1H,

CH

2 Ph), 4.41-4.47 (2d, 2H, CH 2 Ph), 4.81 (d, 1H, J=3.6 Hz, H-I'), 4.63-4.94 (m, 11H, CH 2 Ph), 7.08-7.36 (m, 35H, ArH) . Compound 12 L0 Compound 11 (42 mg, 0.042 mmol, 1 equiv) was dissolved under Argon in a 1:1 mixture of methylene chloride/acetonitrile (1 mL) then treated with dibenzyl N,N-diisopropyl phosphoramidite (42 mL, 0.095 mmol, 2.2 equiv) and tetrazol (13.4 mg, 0.189 mmol, 4.5 equiv). 5 After 1.5 h at room temperature, the mixture was treated at 0 0 C with tert-butyl hydroperoxide. After 40 minutes, the solvents were evaporated and the residue purified by column chromatography (hexane-ethyl acetate 3:1) to give pure 12 (48 mg, 91%). 'H NMR (CDCl 3 , 500MHz): d 3.10 (dd, 0 1H, J=1.7 Hz, J=11.0 Hz, H-6'), 3.36 (dd, 1H, J=2.4 Hz, J=10.9 Hz, H-6'), 3.51 (dd, 1H, J=3.6 Hz, J=10.1 Hz, H-2'), 3.70-3.79 (m, 4H, H-4', H-3, H-4, H-5), 3.82 (Yt, 1H, J=9.8 Hz, J=9.3 Hz, H-3'), 4.03-4.07 (m, 2H, H-2, H-5'), 4.15 (t, 1H, J=3.6 Hz, H-6), 4.28 (d, 1H, CH 2 Ph), 5 4.45-5.01 (m, 9H, CH 2 Ph, H-1, H-i'), 7.10-7.40 (m, 45H, ArH). 31 P NMR (CDCl 3 , 202MHz): d -2.18. Compound C4 Compound 12 (57 mg, 0.045 mmol) was dissolved in methanol 0 (5.3 mL) and buffer sodium acetate-acetic acid pH 5 (5.3 mL) 10% Palladium on charcoal (70 mg) was added and the mixture was stirred overnight under a hydrogen atmosphere. The reaction mixture was filtered over Celite and evaporated. The residue was dissolved in WO 00/32615 PCT/GB99/03715 22 water, filtered over a Sep-pack C-18 and passed through a column of Sephadex G-10 (EtOH-H 2 0 10%) to give C4 (13.8 mg, 73%). 1 H NMR (D 2 0, 500MHz): d 3.41 (dd, 1H, J=3.7 Hz, J=10.7 Hz, H-2'), 3.56-3.65 (m, 2H, H-4', H-4), 3.72-3.76 5 (m, 2H, H-2, H-3), 3.85 (dd, 1H, J=4.4 Hz, J=12.5 Hz, H-6'), 3.89 (dd, 1H, J=2.5 Hz, J=12.5 Hz, H-6'), 3.95 (dd, 1H, J=9.2 Hz, J=10.6 Hz, H-3'), 4.02 (dd, 1H, J=3.4 Hz, J=10.4 Hz, H-5), 4.11 (ddd, 1H, J=2.5 Hz, J=4.2 Hz, J=10.0 Hz, H-5'), 4.31 (t, 1H, J=3.8 Hz, H-6), 4.51-4.54 0 (m, 1H, H-1), 5.40 (d, 1H, J=3.7 Hz, H-I'). 3 P NMR (D 2 0, 202MHz): d +3.00. Compound C4: OH HO 0 HO 3 H3 O HO HO HOO HO O SOH 0 )o WO00/32615 PCT/GB99/03715 23 References: The references mentioned herein are all expressly incorporated by reference. 5 Rademacher et al, Brazilian J. Med. Biol. Res., 27:327 341, 1994. Caro et al, Biochem. Molec. Med., 61:214-228, 1997. 10 Kunjara et al, In: Biopolymers and Bioproducts: Structure, Function and Applications, Ed Svati et al, 301-305, 1995. Zapata et al, Carbohydrate Res., 264;21-31, 1994. 15 Jaramillo et al, J. Org. Chem, 59:3135-3141, 1994. Taglafierri et al, Carbohydrate Res., 266:301-307, 1995. ?0 Vasella et al, 1991, Helf. Chim. Acta, 74:2033-2037, 1991. WO98/11116, WO98/11117 and WO99/38516 (Rademacher Group Limited). .5 0

Claims (23)

1. A method of producing a glycosyl acceptor from chiro-inositol, the method comprising: (a) protecting trans-diequatorial hydroxyl groups 5 of chiro-inositol at positions 2, 3, 4 and 5 to produce a trans-diaxial diol intermediate represented by the general formula; OH PO PO PO OH 0 wherein P is a protecting group; (b) reacting the intermediate of step (a) to produce an epoxide between hydroxyl groups at positions 1 and 6 represented by the general formula; and, 5 PO PO PP (c) transdiaxially opening the epoxide with an alcohol having the formula R-OH to produce the glycosyl acceptor represented by the general formula; 0 PO OH P POOR WO00/32615 PCT/GB99/03715 25

2. The method of claim 1, wherein the starting material is D-chiro-inositol.

3. The method of claim 1, wherein the starting material 5 is L-chiro-inositol.

4. The method of claims 1 to 3 which further comprises the step of protecting the hydroxyl group at position 6. LO

5. The method of any one of claims 1 to 4, wherein the protecting groups P at positions 2, 3, 4 and 5 are bivalent.

6. The method of claim 5, wherein the protecting groups 5 are tetraisopropyldisiloxane (TIPDS).

7. The method of claim 6, wherein the chiro-inositol protected by TIPDS groups is produced by reacting chiro inositol with TIPDS chloride in dimethylformamide with a 0 catalytic amount of 4-dimethylaminopyridine.

8. The method of claim 6 or claim 7, wherein the method comprises the step of further reacting the glycosyl acceptor of step (c) to substitute the protecting groups 5 at positions 2, 3, 4 and 5 for benzyl (Bn).

9. The method of any one of the preceding claims, wherein the alcohol used to trans-diaxially open the epoxide in step (c) is allyl alcohol. 0

10. A method of producing a chiro-inositol containing oligosaccharide comprising: reacting the glycosyl acceptor of any one of the preceding claims with a glycosyl donor to produce the 5 chiro-inositol containing oligosaccharide. WO00/32615 PCT/GB99/03715 26

11. The method of claim 10, wherein the glycosyl donor is derivatised with trichloroacetimidate so that it reacts with the hydroxyl group at position 6 of the glycosyl acceptor to produce the chiro-inositol 5 containing oligosaccharide.

12. The method of claim 11, wherein the glycosyl donor is derivatised at position 1. 10

13. The method of claim any one of claims 10 to 12 which comprises the further step of removing the allyl group at position 1 of the glycosyl acceptor to produce a hydroxyl group. 15

14. The method of claim 13, wherein the allyl group is removed using PdCl 2 /AcOH.

15. The method of any one of claims 10 to 14, wherein the major product of the reaction is the a-configured 20 glycoside.

16. The method of any one of claims 10 to 14, wherein the major product of the reaction is the -configured glycoside. 25

17. The method of any one of claims 10 to 16, further comprising the step of phosphorylating the glycosyl acceptor at position 1. 30

18. The method of any one of claims 10 to 17 comprising the further step of deprotecting the chiro-inositol containing oligosaccharide.

19. The method of any one of claims 10 to 18, wherein 35 the glycosyl donor is a di-, tri, tetra- or WO00/32615 PCT/GB99/03715 27 pentasaccharide.

20. The method of any one of claims 10 to 19, wherein the glycosyl donor is selected from D-galactosamine, D 5 mannosamine or D-glucosamine or a derivative thereof.

21. The method of any one of claims 10 to 20, wherein chiro-inositol containing oligosaccharide is 1D-6-O-(2 amino-2-deoxy-a-D-glucopyranosyl)-chiro-inositol 1 10 phosphate.

22. A method of producing a glycosyl acceptor from chiro-inositol, the method comprising: (a) protecting the trans-diequatorial hydroxyl 15 groups of chiro-inositol at positions 2, 3, 4 and 5 with tetraisopropyldisiloxane (TIPDS); (b) reacting the protected chiro-inositol from step (a) to produce an epoxide between positions 1 and 6; (c) transdiaxially opening the epoxide with an ?0 alcohol having the formula R-OH to produce the glycosyl acceptor having the general formula; and, (d) substituting in one or more steps the TIPDS protecting groups for benzyl (Bn) to produce a glycosyl acceptor having the formula: .5 BnO OH BnO Bn BnOO WO 00/32615 PCT/GB99/03715 28

23. An intermediate represented by the formula: OH TIPDS OH 0 0 0 TIPDS0 OMOM TIPDS- O 0O O AcO OMOM AcO AcO AcOO BnO OMOM BnO Bn BnO O 0