BACKGROUND [0002] Compounds of general formula (I) possess a valuable biological effect, namely they reduce the bronchial hyper-responsiveness, therefore, they can be used as potential active ingredients of pharmaceutical compositions for preventing, treating or alleviating the symptoms of acute and chronic inflammatory disorders of the airways of mammals, including asthma and asthma-related pathologies.

[0003] It was known from the literature (see e. g. Ahmed, T., et al., WO 02/083700), that the mixture of polysulfated glycosides of general formula (I) (I) can be prepared by nitrous acid treatment of such natural products as for example heparin or heparan sulfate, followed by reduction with borohydride and subsequent sulfation of the partially purified samples. The synthesis of the single isomers of general formulas (IA) and (IB) has not been described in the literature.

SUMMARY OF THE INVENTION [0004] The invention provides a new, synthetic method for the synthesis of pure polysulfated glycosides of general formulas (IA) and (IB), having well-defined chemical structures.

[0005] It has been found that compounds of general formula (IX) and (XII)-which can be synthesized by stereospecific synthesis from glucose and glucose amine, and glucuronic acid and glucose amine respectively, can be transformed into compounds of general formulas (IA) and (IB), having well-defined chemical structures. O O MO3SO < OSO3M MO3SO < OSO3M M'OOC,, O O OSO3M M'OOC O O OS03M MO3SO Y OSO3M MO3SO X OSO3M OSO3M OSO3M (IA) (IB) [iduronide] [glucuronide] [0006] Accordingly, the invention provides a process for the synthesis of polysulfated glycosides of general formulas (IA) and (IB), wherein M'means a hydrogen atom, alkali metal or alkali-earth metal atom and M represents an alkali metal or alkali-earth metal atom, which comprises sulfating either a) a glycoside of the general formula (IX), wherein M'means a hydrogen atom, alkali metal or alkali-earth metal atom, to obtain a compound of general formula (IA), or b) a glycoside of the general formula (XII), wherein Rx represents a hydrogen atom, a C14 alkyl group or alkali metal or alkali-earth metal atom, to obtain a compound of general formula (IB), then transforming the sulfated product into a salt, and if desired, hydrolizing the ester group of the carboxylic acid of the so obtained product with base, and, if desired, liberating the compounds of general formulas (IA) or (IB), wherein M'means a hydrogen atom, from the compounds of the general formula (IA) or (IB) formed, wherein M'represents an alkali metal or alkali-earth metal atom.

[0007] Also disclosed are uronic acid glycosides of the general formula (IX), (IX) wherein M'represents a hydrogen atom, alkali metal or alkali-earth metal atom and the use of the compound for the synthesis of an uronic acid glycoside of the general formula (IA), wherein M'represents hydrogen atom, alkali metal or alkali-earth metal and M means an alkali metal or alkali-earth metal atom.

[0008] Also disclosed are uronic acid glycosides of the general formula (XII), (Xll) wherein Rx represents a C, alkyl group, hydrogen atom, alkali metal or alkali-earth metal atom, and the use of the compound for the synthesis of an uronic acid glycoside of the general formula (IB), wherein M'represents hydrogen atom, alkali metal or alkali-earth metal and M means an alkali metal or alkali-earth metal atom.

BRIEF DESCRIPTION OF THE DRAWINGS [0009] A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same become better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein: [0010] Fig. 1 is a graphical representation of a possible synthesis of a compound of general formula (IX) described in detail in the Examples.

[0011] Fig. 2 is a graphical representation of a possible synthesis of a compound of general formula (XII) described in detail in the Examples.

DETAILED DESCRIPTION OF THE INVENTION [0012] The patents, published applications, and scientific literature referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.

[0013] As used in this specification, whether in a transitional phrase or in the body of the claim, the terms"comprise (s)" and"comprising"are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases"having at least"or"including at least". When used in the context of a process, the term"comprising"means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound or composition, the term"comprising"means that the compound or composition includes at least the recited features or components, but may also include additional features or components.

[0014] As used in this specification, the singular forms"a,""an"and"the" specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise.

[0015] The term"about"is used herein to mean approximately, in the region of, roughly, or around. When the term"about"is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term"about"is used herein to modify a numerical value above and below the stated value by a variance of 20%.

[0016] As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value of the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value of the numerical range, including the end-points of the range. As an example, a variable which is described as having values between 0 and 2, can be 0,1 or 2 for variables which are inherently discrete, and can be 0.0, 0.1, 0.01, 0.001, or any other real value for variables which are inherently continuous.

[0017] In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. As used herein, unless specifically indicated otherwise, the word"or"is used in the"inclusive"sense of "and/or"and not the"exclusive"sense of"either/or." [0018] Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of synthetic chemistry include Methods in Carbohydrate Chemistry I. Editors: R. L. Whistler and M. L. Wolfrom. Volume I, Academic Press, New York, London 1962; Methods in Carbohydrate Chemistry II. Editors: R. L. Whistler and M. L.

Wolfrom. Volume II, Academic Press, New York, London 1963; Methods in Carbohydrate Chemistry VI. Editors: R. L. Whistler and J. N. BeMiller. Volume VI, Academic Press, New York, London 1976; and Methods in Carbohydrate Chemistry VII. Editors: R. L. Whistler and J. N. BeMiller. Volume VII, Academic Press, New York, San Francisco, London 1976.

[0019] Reference is made hereinafter in detail to specific embodiments of the invention. While the invention will be described in conjunction with these specific embodiments, it will be understood that it is not intended to limit the invention to such specific embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail, in order not to unnecessarily obscure the present invention.

[0020] Abbreviations used in the description: Ac = acetyl Bz = benzoyl Bn = benzyl Me = methyl Ph = phenyl NIS = N-iodosuccinimide TfOH = trifluoromethanesulfonic acid TBDMSi = tert.-butyl-dimethylsilyl TEMPO = 2,2, 6, 6-tetramethylpiperidin-1-yloxy [0021] An aspect of the invention provides a process for the synthesis of polysulfated glycosides of the general formulas (IA) and (IB), 0 0 M03S0 OSO,3M M03S0 OS03M M'OOC,, O Y O OSO3M M'OOC O O OSO3M M03S0 OS03M M03S0 OS03M OSO3M OSO3M (IA) (IB) [giucuronide] wherein M'stands for a hydrogen atom, alkali metal or alkali-earth metal atom and M represents an alkali metal or alkali-earth metal atom, which comprises sulfating either a) a glycoside of general formula (IX), wherein M'stands for a hydrogen atom, alkali metal or alkali-earth metal atom, to obtain a compound of the general formula (IA), or b) a glycoside of general formula (XII), wherein R'represents a hydrogen atom, a Cl 4 alkyl group or alkali metal or alkali-earth metal atom, to obtain a compound of the general formula (IB), and subsequently transforming the sulfated product into a salt, and if desired, hydrolizing the ester group of the carboxylic acid of the obtained product with base, and, if desired, liberating the compounds of the general formula (IA) or (IB), wherein M'means a hydrogen atom, from the compounds of the general formula (IA) or (IB) formed, wherein M'represents an alkali metal or alkali-earth metal atom.

[0022] According to some embodiments of the process of the invention, compounds of general formula (IA) are synthesized by sulfating a glycoside of general formula (IX) (wherein M'is as described above) in a polar, aprotic solvent with sulfur trioxide or an adduct thereof formed with an organic base (for example, triethylamine or pyridine) or with a non-basic compound (for example, dimethylformamide), using the adduct in excess, at a temperature below 0 °C. The excess of the sulfating agent is decomposed, the formed sulfate ions are removed, and the obtained polysulfated glycosides, if desired after purification by cation exchange resin, are isolated as salts.

[0023] According to other embodiments, compounds of the general formula (IB) are synthesized by sulfating a glycoside of the general formula (XII) (wherein Rx is as described above) according to the method described above, and when Rx represents a C14 alkyl group, the ester group is hydrolyzed by base at a pH of about pH 8.0 to about pH 9.0.

[0024] Compounds of the general formula (IX) and (XII) used as starting materials in the process of the invention are new. They can be synthesized by methods known in organic chemistry. One process for their synthesis is described below in connection with the synthesis of a representative of each of the above general formulas (see also Scheme 1 and 2, Figures 1 and 2 respectfully) by the methods described in detail in the Examples.

[0025] Compounds of general formula (IX) wherein M'represents a hydrogen atom, alkali metal or alkali-earth metal atom, can be synthesized, for example, by glycosylating a donor molecule of the general formula (IV) wherein R represents an acetyl group, which can be obtained from a known compound of the general formula (II) wherein R means an acetyl group and (X) represents an acetoxy group, either in three steps, or directly from the known compound of formula (III) (111) with a known acceptor molecule of formula (V) in the presence of an appropriate activator. The ether type protecting groups are cleaved from the so obtained compound of formula (VI) and the terminal free hydroxyl group of the compound of formula (VII) formed is protected by silylation. The free hydroxyl groups of the so obtained compound of general formula (VIII) wherein R'represents a silyl group and R2 stands for hydrogen atom, are protected by benzoylation, then the silyl group is removed by selective hydrolysis from the formed compound of general formula (VIII), wherein R'represents a silyl group and R2 means a benzoyl group, and the obtained compound of general formula (VIII), wherein R1 represents a hydrogen atom and R2 means a benzoyl group, is transformed by oxidation into compound of general formula (IX), wherein Rl represents a hydrogen atom, R2 means a benzoyl group and R3 stands for an acetyl group, and after removing the protective groups the compound of the general formula (IX), wherein R'means sodium atom and R2 and R3 represents a hydrogen atom, is obtained.

[0026] Compounds of the general formula (XII) wherein Rx represents a C 1-4 alkyl group, hydrogen atom, alkali metal or alkali-earth metal atom, can be synthesized, for example, by glycosylating the known donor molecule of formula (X) with the known acceptor molecule of formula (V) in the presence of an appropriate activator, then the so obtained compound of the general formula (XI) wherein R represents a hydrogen atom is transformed by benzoylation into a compound of the general formula (XI) where R represents a benzoyl group, and the latter is deacylated to give the compound of formula (XII). The methyl ester of formula (XII) is hydrolyzed by base, and if desired the carboxylic acid is liberated from the obtained carboxylic acid salt with acid.

[0027] In the above glycosylation reactions BF3 etherate, NIS, TfOH and the mixture of the latter two can be used as activator. The cleavage of the protective groups can be carried out by acid hydrolysis or reduction in the presence of a catalyst (in the case of ethers and acetals), while in the case of esters Zemplen's method (base catalysed trans-esterification) or hydrolyzis in the presence of a base can be used.

[0028] Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention. However, preferred materials and methods are described. Materials, reagents and the like to which reference are made in the following description and examples are obtainable from commercial sources, unless otherwise noted.

[0029] The following examples are intended to further illustrate certain preferred embodiments of the invention and are not limiting in nature. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein.

EXAMPLES [0030] The Rf values given in the examples were determined by thin layer chromatography using silica gel (DC-Alufolien Kieselgel 60 F254, Merck, Darmstadt) and the following mixtures of solvents: (A) Ethyl acetate (B) Ethyl acetate-hexane 1: 1 (C) Ethyl acetate-hexane 1: 2 (D) Ethyl acetate-hexane 1: 3 (E) Ethyl acetate-hexane 1: 5 (F) Ethyl acetate-hexane 2: 1 (G) Ethyl acetate-hexane 5: 1 (H) Ethyl acetate-ethanol 3: 1 (I) Ethyl acetate-ethanol 5: 1 [0031] The spots were detected either in UV light or by spraying the plates with a 1: 1 mixture of 0.1 M KMn04-1 M H2SO4 followed by heating to 200 °C. Column chromatography was performed on Kieselgel 60. Optical rotations were measured at 20 °C. NMR spectra were recorded with Bruker Avans 500 MHz spectrometer using Me4Si as the internal standard. The assignments of the protons were based on homonuclear decoupling and DNOE experiments. Multiplicities of the 13C spectra were obtained from DEPT experiments. Connectivities between identified protons and protonated carbons were observed by means of HETCOR experiments.

[0032] In the case of acylation reactions carried out in the presence of pyridine the term"work-up"means that if the product was not crystalline after pouring the reaction mixture into ice-water, it was extracted with an organic solvent, the organic layer was washed sequentially with water, 1 M ice-cold aqueous sulfuric acid solution until permanent acidity, water, 5% aqueous sodium bicarbonate solution and water, dried, filtered and the solvent evaporated in vacuum.

Example 1 [0033] 2, 5-Anhydro-l, 4, 6-tri-0-sulfato-3-(2, 3, 4-tri-0-sulfato-ß-D- idopyranuronido)-D-mannitol hepta sodium salt (IA, M'=M= Na) [0034] 30 g (48%, 175 mmol) of sulfur trioxide-dimethylformamide complex was suspended in 50 ml of dry dimethylformamide with stirring and the mixture was cooled to-20 °C and 5.0 g (14 mmol) of 2, 5-anhydro-3-O-(a-L-idopyranosyl uronic acid)-D- mannitol sodium salt (IX, M'= Na) was gradually added at a rate to keep the temperature below-15 °C. After 15 min, the temperature of the mixture was raised to- 5 °C and kept there for about 45 min.

[0035] Thereafter the reaction mixture was poured into a stirred and cooled (0 °C) solution of 28 g of sodium bicarbonate and 350 ml of water. The solution was neutralized with 1 M sulfuric acid, concentrated and the residue was filtered and washed with methanol. The solid residue was dissolved in 250 ml of water and M aqueous strontium acetate solution was added until no more precipitate (SrSO4) was formed.

The precipitate was filtered off and the filtrate was submitted to a column loaded with CHELX 100 resin (sodium form) (10 mL) in order to remove strontium ions. The column was eluted with distilled water and the eluate was concentrated. The residue was treated with ethanol, filtered and washed with ethanol to yield 12 g (88%) of the title compound; [a] D-5° (c 1, H20) ;'HNMR (D20) 8 : 5.29 (m, 1H, H-l'), 5.17 (m, 1H, H-2'), 4.85 (m, 1H, H-4') 4.83 (t, H-4), 4.78 (m, 1H, H-5'), 4,60 (dd, IH, H-3), 4.56 (m, 1H, H-3'), 4.55 (dt, 1H, H-5), 4.50 (dt, 1H, H-2), 4.32 (dd, 1H, Ha-6), 4.26 (dd, IH, Ha- 1), 4.25 (dd, 1H, Hb-6), 4.22 (dd, 1H, Hb-1); Juge" 11. 2, Jla, 2 4. 0, Jlb, 2 6. 5, J2, 3 3. 9, J3, 4 3.1, J4,5 3. 1, J5,6a 3. 4, J5, 6b 8. 3, J6gem 11.2, J1',2'#3,J2',3'#3,J3',4'#3,J4',5'#3 Hz. 13C NMR (D20) 8 : 176.5 (C-6'), 100.1 (C-1'), 84.7 (C-4), 84.2 (C-5), 83.3 (C-2), 83.3 (C-3) 74.6 (C-4') 73.2 (C-3') 73.0 (C-2') 70.1 (C-6), 69.6 (C-1), 69.3 (C-5'); Cl2Hl3029S6Na7 Calculated: C 14.79 ; H 1.34 ; Na 16.51 ; S 19.74. Found: C 14.43 ; H 1.98 ; Na 16.40 ; S 19.45.

[0036] The starting material-2, 5-anhydro-3-O-(a-L-idopyranosyl uronic acid)-D- mannitol sodium salt can be synthesized, for example, by the following method (the reaction pathway shown in Scheme 1, Fig. 1) : [0037] Step a) [0038] Phenyl-2, 4, 6-tri-O-acetyl-3-0-benzyl-1-thio-L-idopyranoside (II, R = Ac, X = SPh) [0039] 40 g (91 mmol) of crude tetraacetate (II, R = Ac, X = OAc) [C. A. A. van Boeckel, T. Beetz, J. N. Vos, A. J. M. de Jong, S. F. van Aelst, R. H. van den Bosch, J. M. R.

Mertens, F. A. van der Vlugt, J. Carbohydr. Chem. 4 (1985) 293-321] was dissolved in 500 ml of dichloromethane and 11 ml (107 mmol) of thiophenol and 31 ml (245 mmol) of BF3. Et2O were added to the stirred solution at 0 °C. Stirring was continued at room temperature for 90 min, then the mixture was washed with 5% aqueous sodium bicarbonate solution and water. The dried solution was concentrated to yield 46 g of crude product, which was purified by column chromatography using eluent (solvent D).

[00401 Concentration of the first fraction (Rf 0. 6, solvent C) gave 5.6 g (13%) of the p-anomer, [a] D +27° (c 1, CHC13).

[0041] Concentration of the second fraction (Rf 0.5, solvent C) gave 28 g (63%) of the a-anomer, [a] D-95° (c 1, CHC13). The'H-NMR spectrum was identical with the one described in the literature [C. A. A. van Boeckel, T. Beetz, J. N. Vos, A. J. M. de Jong, S. F. van Aelst, R. H. van den Bosch, J. M. R. Mertens, F. A. van der Vlugt, J. Carbohydr.

Chem. 4 (1985) 293-321].

[0042] Step b) [0043] Phenyl-3-0-benzyl-1-thio-a-L-idopyranoside (II, R = H, X = SPh) [0044] 6.4 g of the triacetate obtained in Step a) was dissolved in 70 ml of methanol and 0.5 ml of 2M methanolic sodium methylate solution was added to the stirred solution at room temperature. After 2 h the mixture was neutralized with solid carbondioxide, concentrated and the residue was purified by column chromatography using eluent (solvent B) to yield 3.4 g (71.5%) of the title compound. [a] D-151° (c 1, CHC13), Rf 0.2 (solvent B).

[0045] Step c) [0046] Phenyl-3-0-benzyl-4, 6-0-benzylidene-1-thio-a-L-idopyranoside (IV, R = H) [0047] 10 g of the thiophenyl glycoside obtained in Step b) was dissolved in 100 ml of dimethylformamide, 50 mg of p-toluenesulfonic acid and 10 ml of benzaldehyde dimethylacetal were added to the solution and the mixture was stirred at 70 °C for 5 h under vacuum (10 Kp). Then the solution was cooled, diluted with ether, washed with 5% aqueous sodium bicarbonate solution and water, dried and concentrated. The residue was purified by column chromatography using eluent (solvent E) to yield 8 g (64%) of the title compound. [a] D-145° (c 1, CHCl3), Rf 0. 55.

[0048] When the anomeric mixture of thioglycosides was used as starting material, besides the above mentioned a-anomer (46%) a small amount of (3-anomer (8%) can also be isolated from the mixture by column chromatography; [a] p +80° (c 1, CHC13), Rf 0. 4.

[0049] Step d) [0050] Phenyl-2-0-acetyl-3-0-benzyl-4, 6-0-benzylidene-1-thio-a-L-idopyranoside (IV, R = Ac) [0051] 3.5 g (7.8 mmol) of the thiophenyl glycoside obtained in Step c) was dissolved in 10 ml of pyridine, 5 ml of acetic anhydride was added and the mixture was left at room temperature overnight. After work-up, the obtained crude product was recrystallized from 1.5 fold ethanol to yield 2.5 g of the title compound. Column chromatography (solvent E) of the mother liquor gave further 0.6 g of the title compound, this way the combined yield was 80%. Mp 120-122 °C, [a] D-123° (c 1, CHCI3), Rf 0. 4.

[0052] The title compound can also be obtained by the following method: 13.5 g (30.5 mmol) of the known [Ch. Tabeur, F. Machetto, J. -M. Mallet, P. Duchaussoy, M.

Petitou, P. Sinay, Carbohydr. Res., 281 (1996) 253-276] 1, 2-di-O-acetyl-3-O-benzyl- 4, 6-O-benzylidene-L-idopyranose of formula (III) was dissolved in 240 ml of dichloromethane, 4 ml (36 mmol) of thiophenol and 10 ml (79 mmol) of BF3. Et2O were added at-20 °C and the reaction mixture was stirred at this temperature for 30 min, then at-10 °C for 45 min. The reaction mixture was cooled to-20 °C, 17 ml of Et3N was added and the temperature was allowed to rise to room temperature. The so obtained solution was washed with water, dried and concentrated. The residue was recrystallized from 1.5 fold ethanol to yield 5.5 g of the title compound, identical with the one obtained by the above procedure. Column chromatography of the mother liquor gave further 23 g of the title compound, this way the combined yield was 50%.

[0053] Step e) [0054] 3-0- (2-0-acetyl-3-0-benzyl-4, 6-O-benzylidene-a-L-idopyranosyl)-2, 5-<BR> anhydro-1, 6-di-0-benzoyl-D-mannitol (VI) [0055] 6.2 g (12.6 mmol) of thioglycoside obtained in Step d) and 5.6 g (15 mmol) of 2, 5-anhydro-1, 6-di-O-benzoyl-D-mannitol of formula (V) [D. A. Otero and R.

Simpson, Carbohydr. Res., 128 (1984) 79-86; N. Barroca and J-C. Jacquinet, Carbohydr. Res., 337 (2002) 673-689] were dissolved in 180 ml of dry dichloromethane, 18 g of freshly heated molecular sieve (4A) was added to the stirred solution and stirring was continued at room temperature for 30 min. Then the mixture was cooled to-40 °C, 5.6 g (25 mmol) of NIS and 0.5 ml of TfOH were added. Stirring was continued at this temperature for 20 min, then 1 ml of Et3N was added and the temperature was allowed to rise to room temperature. The reaction mixture was filtered, the filtrate was diluted with dichloromethane, washed with aqueous sodium thiosulfate solution, aqueous sodium bicarbonate solution and subsequently with water.

The dried organic solution was concentrated and the residue was purified by column chromatography (solvent D then C) to yield 7.7 g (81%) of the title compound as syrup; [a] D-28° (c 1, CHC13), Rf 0. 3 (solvent C).

[0056] Step f) [0057] 3-0- (2-0-acetyl-a-L-idopyranosyl)-2, 5-anhydro-1, 6-di-O-benzoyl-D- mannitol (VII) [0058] 4.7 g (6.2 mmol) of the product of formula (VI) obtained in Step e) was dissolved in a mixture of 150 ml of methanol and 15 ml of water, then 0.5 ml of acetic acid and 0.5 g of 10% Pd/C catalyst were added. The reaction mixture was shaken in hydrogen atmosphere for 6 h, when according to TLC hydrogenolyzis of both the benzyl and the benzylidene group was complete (Rf 0. 9-> 0.3, solvent A). The mixture was filtered, the filtrate was concentrated, toluene was added and evaporated from the residue and the so obtained crude product was recrystallized from a mixture of ethyl acetate-hexane to yield 3.44 g (95%) of the title compound; Mp 142-143 °C, [a] D-13° (c 1, CHCI3).

[0059] Step g) [0060] 3-0- (2-0-acetyl-3, 4-di-O-benzoyl-6-0-tert-butyldimethylsilyl-a-L- idopyranosyl)-2, 5-anhydro-1, 2, 6-tri-0-benzoyl-D-mannitol (VIII, Rl = TBDMSi, R2 = Bz) [0061] 11.4 g (20.14 mmol) of the product of formula (VII) obtained in Step f) was dissolved in 70 ml of pyridine and 3.65 g (24 mmol) of tert-butyldimethylsilyl chloride was added at 0 °C to the stirred solution. Stirring was continued at 20 °C for 2 h, then further 1 g (6.6 mmol) of tert-butyldimethylsilyl chloride was added. The reaction mixture was kept at room temperature overnight, when the sylilation reaction was complete (Rf 0. 2 ~ 0.7, solvent A). The reaction mixture was cooled to 0 °C and 8.75 ml (75.5 mmol) of benzoyl chloride was added over 10 min. The temperature of the reaction mixture was allowed to rise to room temperature and after 2 h the mixture was poured onto ice, extracted with dichloromethane and after ususal work-up the solvent was evaporated and the residue was purified by column chromatography (solvent C).

The so obtained syrupy crude product (20 g, 100%) was used in the next step without further purification; Rr 0. 7, [a] D-55° (c 1, CHC13).

[0062] Step h) <BR> <BR> <BR> <BR> [0063] 3-0- (2-0-acetyl-3, 4-di-O-benzoyl-a-L-idopyranosyl)-2, 5-anhydro-1, 4, 6-tri- 0-benzoyl-D-mannitol (VIII, Rl = H, R2 = Bz) [0064] 20 g (20.36 mmol) of the crude product obtained in Step g) was dissolved in a mixture of 300 ml of methanol and 300 ml ethanol, and 40 ml of M sulfuric acid was added to the stirred solution at room temperature. After 30 min further 20 ml of M sulfuric acid was added and stirring was continued for 5 h, when according to TLC the cleavage of the silyl group was complete (Rf 0. 7 ~ 0. 4, solvent C). The pH of the solution was adjusted to 5 by addition of solid sodium bicarbonate, the reaction mixture was filtered and the filtrate was concentrated. The residue was dissolved in dichloromethane, washed with 5% aqueous sodium bicarbonate solution and water, dried and concentrated. The residue was purified by column chromatography (solvent C) to yield 17 g (97%) of the title compound as syrup; Rf 0. 4, [a] D-61° (c 1, CHCI3).

[0065] Step i) [0066] 2, 5-Anhydro-1,4,6-tri-O-benzoyl-3-O-(2-O-acetyl-3,4-di-O-benzo yl-α-L- idopyranosyl uronic acid)-D-mannitol (IX, Rl = H, R2 = Bz, R3 = Ac) [0067] 13.85 g (15.65 mmol) of the product obtained in Step h) was dissolved in 100 ml of dichloromethane, 50 mg of TEMPO, 260 mg of KBr, 400 mg of Bu4NBr and 100 ml of 4% aqueous sodium bicarbonate solution were added to the stirred solution.

The reaction mixture was cooled to 0 °C and a mixture of 180 ml of 0.6 M aqueous NaOCI solution, 25 ml of 4% aqueous sodium bicarbonate solution and 50 ml of saturated aqueous sodium chloride solution was added dropwise over a period of 45 min. The organic layer was separated, the water layer was extracted with dichloromethane, and the combined organic layers were washed with water, dried and concentrated. The residue was purified by column chromatography using solvent (F) first for eluting the faster running by-products, then solvent (I) for eluting the main product. Concentration of the latter fractions (Rf 0. 85) gave 10.65 g (74.4%) of the title compound as amorphous foam, which was according to NMR spectroscopy a-1 : 3 mixture of the free carboxylic acid and its sodium salt. [a] D-61° (c 1, CHCI3).

[0068] Step j) [0069] 2, 5-Anhydro-3-O-(α-L-idopyranosyl uronic acid)-D-mannitol sodium salt (IX, R =Na, RZ=R3 =H) [0070] 2.25 g (2.5 mmol) of the product obtained in Step i) was dissolved in 50 ml of methanol and 1.5 ml of 2 M methanolic sodium methylate solution was added to the stirred solution at room temperature. After 30 hours, the solution was treated with a cation exchange resin (H) to remove sodium ions, filtered and concentrated. The residue was dissolved in water and extracted with chloroform to remove methyl benzoate, the pH of the aqueous solution was adjusted to 8 with M NaOH to give after freeze-drying the sodium salt. The solid salt was filtered with ethyl acetate to yield 0.7 g (82.5%) of the title compound. [a] p 0° (c 1, water), the free carboxylic acid has an optical rotation of [a] D-12° (c 1, water).

Example 2 [0071] 2, 5-Anhydro-1, 4, 6-tri-0-sulfato-3-(2, 3, 4-tri-0-sulfato-ß-D- idopyranuronido)-D-mannitol hepta potassium salt (IA, M'=M= K) [0072] 3.7 g (48%, 22 mmol) of sulfur trioxide-dimethylformamide complex was suspended in 5 ml of dry dimethylformamide with stirring, the mixture was cooled-20 °C and a solution of 0. 71 g (2 mmol) of 2,5-anhydro-1, 4, 6-tri-O-sulfato-3- (2, 3, 4-tri-0- sulfato-ß-D-idopyranuronido)-D-mannitol hepta sodium salt (prepwered according to the method described in Example 1) in 3 ml of dimethylformamide was added dropwise at such a rate to keep the temperature below-15 °C. Stirring was continued at this temperature for 15 min, then at-5 °C for 45 min. The reaction mixture was cooled to- 20 °C and 1 ml of ethanol was added at such a rate to keep the temperature below-15 °C. Then the reaction mixture was poured into a stirred solution of 5 g potassium acetate in 40 ml of methanol at 0 °C. The precipitated crystals were filtered off and washed with 3x40 ml of methanol. The so obtained crude product was dissolved in 15 ml of water, the pH of the solution was adjusted to 8 with 1 M potassium hydroxide solution and 1 M aqueous strontium acetate solution was added until no more precipitate (SrS04) was formed. The precipitate was filtered off and the filtrate was submitted to a column loaded with CHELX 100 resin (potassium form) (15 mL) in order to remove strontium ions. The column was eluted with dwastilled water and the eluate was concentrated. The solid residue was treated with methanol, filtered, washed with ethanol and dried to yield 1.85 g (85%) of the title compound. [a] D-4° (c 1, water).

C2Hl302sS6K7 Calculated: C 13.26 ; H 1.21 ; K 25. 17; S 17.69. Found: C 13.13 ; H 1.48 ; K 24.98 ; S 17.45 ; Sr 0. 011. According to NMR spectroscopy the sample contains 0.25 equivalent of potassium acetate and 0.07 equivalent of ethanol.

Example 3 [0073] 2, 5-Anhydro-1, 4, 6-tri-O-sulfato-3- (2, 3, 4-tri-O-sulfato-/j-D- glucopyranuronido)-D-mannitol hepta sodium salt (IB, M'=M=Na) [0074] 1.7 g (48%, 10 mmol) of sulfur trioxide-dimethylformamide complex was suspended in 3 ml of dry dimethylformamide with stirring, the mixture was cooled-20 °C and a solution of 0.35 g (1 mmol) of the methyl ester of formula (XII) in 3 ml of dimethylformamide was added dropwise at such a rate to keep the temperature below- 15 °C. Stirring was continued at this temperature for 15 min, then at-5 °C for 45 min.

The reaction mixture was cooled to-25 °C and 0.5 ml of ethanol was added at such a rate to keep the temperature below-15 °C. Then the reaction mixture was poured into a stirred solution of 2 g sodium acetate in 20 ml methanol at-5 °C. The precipitated crystals were filtered off and washed with methanol. The so obtained solid product was dissolved in 5 ml of water, the pH of the solution was adjusted to 8-9 with M sodium hydroxide solution and further 1 ml of M sodium hydroxide solution was added to hydrolyze the methyl ester of the formula (XIII). The solution was kept at room temperature for 15 h, then diluted with 25 ml of water and 1 M aqueous stroncium acetate solution was added until no more precipitate (SrSO4) was formed. The precipitate was filtered off and the filtrate was submitted to a column loaded with CHELX 100 resin (sodium form) (10 mL) in order to remove strontium ions. The column was eluted with distilled water and the eluate was concentrated. The residue was treated with ethanol, filtered and washed with ethanol to yield 0.75 g (79%) of the title compound; [a] D +15° (c 1, H20) ; NMR (D2O) 8 : IH, 4.96 (t, 1H, H-4), 4.92 (dd, 1H, H-4'), 4.91 (d, 1H, H-l') 4.84 (dd, IH, H-3'), 4.12-4. 53 (m, 9H) ; Jl', 2'7 0, J2', 3'3 4, J3',4' 5.3, J4',5' 3.8 Hz. 13C, 173.5 (C-6'), 99.2 (C-1'), 83.6, 81.5, 81.0, 80.3, 77.1, 77.1, 75.6, 74.6 (C-2,3, 4,5, 2', 3', 4', 5') 66.8, 66.6 (C-1,6). C,2HI3029S6Na7 Calculated: C 14.79 ; H 1.34 ; Na 16. 51; S 19. 74. Found: C 14. 58; H 1. 72; Na 16. 44; S 19. 55.

[0075] The starting material of the formula (XII) can be synthesized for example by the following method: [00761 Step a) [0077] 2, 5-Anhydro-1, 6-di-0-benzoyl-3-0-(2, 3, 4-tri-0-acetyl-ß-D- glucopyranosyluronate methyl ester)-D-mannitol (XI, R = H) [0078] 0.74 g (2 mmol) of 2,3, 4-Tri-O-acetyl-ß-D-glucopyranosyluronate methyl ester I-O-trichloroacetimidate of formula (X) [G. N. Bollenback, J. W. Long, D. G.

Benjamin, J. A. Lindquwast., J. Am. Chem. Soc., 77 (1955) 3310-3315] was dissolved in 25 ml of dichloromethane, then 0.96 g (2 mmol) of 2, 5-anhydro-1, 6-di-O-benzoyl-D- mannitol of formula (V) [D. A. Otero azd R. Simpson, Carbohydr. Res., 128 (1984) 79- 86; N. Barroca and J-C. Jacquinet, Carbohydr. Res., 337 (2002) 673-689] and 5 g of freshly heated molecular sieve (4A) were added to the stirred solution and stirring was continued for 2 h at room temperature. The reaction mixture was cooled to-40 °C and 0.1 ml of trimethylsilyl triflate was added. After 5 min the reaction was quenched by the addition of 0.5 ml of triethyl amine, diluted with 25 ml of dichloromethane, washed with 5% aqueous sodium bicarbonate solution and water, dried and concentrated. The residue was purified by column chromatography (solvent B).

[0079] Concentration of the first fraction (Rf 0.8) yields 0.1 g (12%) of 3-0-acetyl- 1, 6-di-O-benzoyl-D-mannitol.

[0080] Concentration of the second fraction (Rf 0.65) yields 0.15 g (11 %) of orthoester as syrup [a] D +12° (c 1, CHCI3).

[0081] Concentration of third fraction (Rf 0.5) yields 0.5 g (36%) of the title compound (XI, R = H), the purity of which was 60 % acording to NMR spectroscopy.

[0082] Step b) [0083] 2, 5-Anhydro-1, 6-di-O-benzoyl-3-0- (2, 3, 4-tri-O-acetyl-/-D- glucopyranosyluronate methyl ester)-D-mannitol (XI, R = Bz) [0084] 1.5 g of the syrup obtained in Step a) was dissolved in 10 ml pyridine and 0.5 ml of benzoyl chloride was added to the stirred solution. The reaction mixture was stirred at room temperature for 1.5 h, then diluted with dichloromethane and concentrated after the work-up. The residue was recrystallized from ethanol to yield 0.9 g (52%) of the title compound. Mp: 140-141 °C, Rf 0. 6 (solvent B) ; [a] D-18° (c 1, CHC13).

[0085] Step c) [0086] 2, 5-Anhydro-3-0- (-D-glucopyranosyluronate methyl ester)-D-mannitol (EI) [0087] 1.2 g of the crystalline compound of formula (XI, R = Bz) obtained in Step b) was suspended in 20 ml of methanol and 0.3 ml of 2 M methanolic sodium methylate solution was added to the stirred mixture. he reaction mixture was stirred at 45 °C for 5 h, then cooled to room temperature, neutralized with ion exchange resin, filtered and the filtrate was concentrated. he residue was dissolved in water and extracted with diethyl ether to remove the formed methyl benzoate. The aqueous layer was concentrated to yield 0.5 g (93%) of the title compound as syrup, Rf 0. 3 (solvent/7). NMR (DMSO) 8 : H, 4.30 (d, 1H, H-1'), 3.99 (t, 1H, H-3), 3.93 (m, 1H, H-4), 3.82 (m, 1H, H-5), 3.70 (d, 1H, H-5'), 3.66 (s, 3H, 6'-OMe), 3.65 (m, 1H, H-5), 3.34-3. 52 (m, 4H, H2-1, 6), 3.31 (t, 1H) H-4'), 3.17 (t, 1H, H-3') 3.00 (t, 1H, H-2') ; J2, 3 3. 8, J3, 4 3. 8, J1',2' 7.8, J2 3 8. 2, J3 4 9. 0, J4 5 9.3 Hz.'3C, 169.8 (C-6'), 102.5 (C-1'), 85.9 (C-3), 84.6 (C-5), 82.9 (C-2), 76.7 (C_4), 76.3 (C-3'), 76.2 (C-5'), 73.3 (C-2'), 71.9 (C-4'), 61.9 (C-l), 61.6 (C-6), 52-1 (6'-OMe)