Background:

Glycosaminoglycans are a class of biomolecules characterized by linear repeating disaccharide units. With the exception of keratan sulfate, the glycosaminoglycans are composed of linearly connected disaccharides containing a uronic acid (either L-lduronic acid or D-glucuronic acid) and a glucosamine moiety. L-lduronic acid is the predominant uronic acid in dermatan sulfate and heparin.

In the biosynthesis of both dermatan sulfate and heparin, it has been established that the iduronic acid moieties are incorporated by C-5 epimerization of the corresponding D-glucuronic acid moieties within the growing polymer chain. That is to say, they are made in situ rather than through the incorporation of UDP- iduronic acid. Thus there is not a cheap natural source of iduronic acid synthons for use in drug discovery and development.

Research into glycosaminoglycan fragments for use in medicine, particularly anticoagulant products has provided a need for iduronic acid synthons. Several preparations of iduronic acid synthons and examples of their use have appeared in the literature. W003/022860 provides a 12 step synthesis of an idose synthon (example 3) which may be used to form a disaccharide and subsequently oxidized to the corresponding iduronic acid containing disaccharide in 4 steps (example 17). WO99/25720 similarly discloses the preparation of a different disaccharide via a protected L-idose donor sugar (scheme 1 and 2).

Other efforts to develop suitably protected iduronic acid synthons have also been reported. Bonnaffe and co-workers disclosed the synthesis of several synthons suitable for building disaccharides (Eur. J. Org. Chem., 2003, 3603-3620 ). Commercially available diacetone glucose was converted to either a trichloroacetimidyl iduronic acid or bromoiduronyl sugar donor in modest yields. Unfortunately both syntheses required the use of low temperatures (-78 degrees) and included several chromatographic steps making them unsuitable for industrial production.

Hansen and co-workers ( J . Org. Chem. 2015, 80, 3777-3789) have reported the conversion of B5 to B6 in three steps and 40% overall yield.

The present invention provides a new approach to the conversion of B5 to B6 in which the transformation is effected in a single pot in overall 80% yield isolation and with purification by precipitation. This new method provides a significant industrial advantage over previous methods.

Similarly, the current invention provides for a one pot conversion of B6 to B9 in comparable yield but greater industrial efficiency than reported by Hansen and co-workers (J. Org. Chem. 2015, 80, 3777-3789) for a similar intermediate in a 2 step process.

B6 B9

In a third aspect of the invention a highly regioselective protection of the 02 hydroxyl and not the 04 hydroxyl of the L-iduronic acid unit in the disaccharide BA2 is introduced. The differentiation between these 2 hydroxyl groups is generally considered difficult and as a critical step for producing an acceptor unit that can be used for elongation. Bonaffe ( Eur . J. Org. Chem., 2003, 3603-3620) discloses the selective acetylation of a disaccharide building block in a two step process in 60% overall yield. The Bonaffe procedure suffers several disadvantages in particular the use of dibutyltin oxide a toxic reagent which is difficult to remove from the reaction product requiring laborious chromatography, and the need to recycle byproducts to achieve an effective yield.

The present invention provides an improved process from BA2 to a selectively protected disaccharide BA3 building block in 83% yield in a single step. The product may be purified by crystallization which offers significant industrial advantages. The disaccharide acceptor BA3 is a key intermediate in the synthesis of the anticoagulant drug Fondaparinux and has been used as such in the prior art ( J . Org. Chem. 2016, 81, 162-184).

Thus the present invention provides new chemical compositions, and methods for their preparation, which are industrially relevant in the manufacture of glycosaminoglycans, particularly Fondaparinux. Summary of invention:

In one embodiment the invention provides for compounds of formula I, useful as intermediates in the synthesis of glycosaminoglycans, glycosaminoglycan fragments, analogs of glycosaminoglycans or glycosaminoglycan fragments or in the development of new medicinal agents. The groups Ri and R ₄ are independently selected from the group consisting of benzoyl or arylacyl; R ₂ is selected from the group C1 to C4 alkyl, benzyl or substituted benzyl; and R ₃ is selected from the group selected from OH, trichloroacetimidoyl or a moiety of formula II; such that when R ₃ is moiety II, R ₅ is benzyloxycarbonylamino or azido, and R ₆ is either an acyl protecting group, selected from acetyl, pivaloyl or a benzoyl group, or a carbonate protecting group selected from benzyloxycarbonate, allyloxycarbonate or fluorenylmethyloxycarbonyl, and the stereochemistry of the center to which R ₃ is attached is of the alpha configuration.

In a further embodiment the invention provides for compounds of formula I wherein Ri and R ₄ are benzoyl; R ₂ is selected from the group C1 to C4 alkyl, benzyl or substituted benzyl; and R ₃ is selected from the group selected from

OH, trichloroacetimidoyl or a moiety of formula II.

In a further embodiment the invention provides for compounds of formula I wherein Ri and R ₄ are benzoyl; R ₂ is selected from the group C1 to C4 alkyl, benzyl or substituted benzyl; and R ₃ is OH. In a further embodiment the invention provides for compounds of formula I wherein Ri and R ₄ are benzoyl; R ₂ is selected from the group C1 to C4 alkyl, benzyl or substituted benzyl; and R ₃ is trichloroacetimidoyl. In a further embodiment the invention provides for compounds of formula I wherein Ri and R ₄ are benzoyl; R ₂ is selected from the group C1 to C4 alkyl, benzyl or substituted benzyl; and R ₃ is a moiety of formula II.

In a further embodiment the invention provides for compounds of formula I wherein Ri and R ₄ are benzoyl; R ₂ is methyl; and R ₃ is OH.

In a further embodiment the invention provides for compounds of formula I wherein Ri and R ₄ are benzoyl; R ₂ is methyl; and R ₃ is trichloroacetimidoyl.

In a further embodiment the invention provides for compounds of formula I wherein Ri and R ₄ are benzoyl; R ₂ is methyl; and R ₃ is a moiety of formula II.

In a further embodiment the invention provides a method of synthesis of a compound of formula I wherein Ri _, R ₂ , R ₃ and R ₄ are H; comprising the step of reacting a compound of formula III i) concentrated aqueous acid HX in a suitable solvent at room temperature, where X is selected from -Cl, -Br, -I, -CI0 ₄ , -HS0 ₄ , - H ₂ P0 ₄ , followed by, ii) dilute aqeuous acid HX in a water miscible solvent at temperatures between room temperature and 100 degrees C.

Preferably, the aqueous acid HX is hydrochloric acid

Preferably, the method of synthesis of a compound of formula I wherein Ri _, R ₂ , R ₃ and R ₄ are H is a method comprising the step of reacting a compound of formula III with: i) concentrated aqueous hydrochloric acid in tetrahydrofuran solvent at room temperature followed by, ii) dilute aqeuous hydrochloric acid in tetrahydrofuran solvent at temperatures between 50 and 70 degrees C.

In a further embodiment the invention provides a method for the preparation of compound of formula I wherein Ri and R ₄ are independently benzoyl or arylacyl; R ₂ is selected from the group C1 to C4 alkyl, benzyl or substituted benzyl; and R ₃ is OH; comprising the steps of reacting a compound of formula I in which Ri _, R ₂ ,

R ₃ and R ₄ are H with: a. methanesulfonyl chloride or toluenesulfonyl chloride and an organic base in an aprotic solvent followed by b. benzoyl chloride or aroylacyl chloride and an organic base followed by c. excess alcohol reagent selected from C1 to C4 alkyl substituted alcohol, benzyl alcohol or substituted benzyl alcohol, such as e.g methanol; wherein the three steps are preferably performed consecutively in a single reaction.

In a further embodiment the invention provides for a method for the preparation of a compound of formula IV wherein R ₄ is selected from the group consisting of acetyl, chloroacetyl, pivaloyl, benzoyl or arylacyl; and R ₂ is selected from the group C1 to C4 alkyl, benzyl or substituted benzyl, R ₅ is benzyloxycarbonylamino or azido; involving the selective acylation process of reacting a compound of formula V with an organic base B and an acylating agent RCOX in an aprotic solvent at a temperature between -100 to 40 degrees C, where X is selected from -Cl, -Br, - O2CR and R is selected from -CH ₃ . -CH ₂ CI, -C(CH ₃ ) ₃ , -Ph, -PhCH ₃ , -PhOMe, - PhN0 ₂ , -PhCI, -PhBr. In relation to the method for the preparation of a compound of formula IV discussed immediately above - a preferred method is a method wherein:

- each instance of R ₂ is methyl and R ₅ is benzyloxycarbonylamino; and/or

- the base B is selected from triethylamine, trimethylamine, diisopropylethylamine, pyridine, lutidine; and/or

- the acylating agent is benzoyl chloride (R: -Ph) and 2-3 equivalents of the reagent is used; and/or

- the reaction is performed at a temperature between 0 to -50 degrees C; and/or

- the selective acylation is effected by reacting a compound of formula V with 2.4 equivalents benzoyl chloride and triethylamine in dichloromethane solvent while the temperature is -10 to -30 degrees C.

In a further embodiment the invention relates to use of a compound according to the invention as described herein (see above and/or any of claims 1 to 5 below) in the synthesis of fondaparinux (preferably fondaparinux sodium).

In a further embodiment the invention relates to use of a method according to the invention as described herein (see above and/or any of claims 6 to 13 below) in the synthesis of fondaparinux (preferably fondaparinux sodium). EXAMPLES:

The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention in any way as many variations and equivalents that are encompassed by the present invention will become apparent to those skilled in the art upon reading the present disclosure.

List of abbreviations:

Ac: Acetyl Bz: Benzoyl Bn: Benzyl

DBU: Diazabicyclo[5.4.0]undec-7-ene (DBU) DCM: Dichloromethane Et: Ethyl Me: Methyl Tf: Trifluoromethanesulfonyl

THF: Tetrahydrofuran TMS: Trimethylsilyl Ts: Tosyl, 4-methylbenzenesulfonyl

Preparation of B6 (3-O-Benzyl-L-iduronic acid): Compound B5 can be prepared according to the methods described by Hansen et. al (1) Org Lett. 2009, vol 11, 20, 4528-4531. 2) J. Org. Chem. 2015, 80, 3777-3789) which also describes the conversion

B5 to B6 in a 3 step procedure. The current 1 pot procedure is superior in yield, purity, simplicity and industrial applicability.

To B5 (61.0 g, 0.20 mol) was added THF (110 ml_), HCI (36%, 220 ml.) and stirred for 4 hrs. Then was added THF (150 ml.) and water (750 ml.) and the mixture heated to 60°C for 16 hrs. After cooling NaHC0 ₃ (203 g, 2.41 mol) was added in portions to pH = 2-3,

NaCI (162 g, 2.8 mol) and THF (240 ml_). The phases were separated, and the aqueous phase extracted with THF (2x250 ml_). The combined organic phase was dried (MgS0 ₄ ), filtered and evaporated to give a brown solid. This solid was purified by stirring with DCM (250 ml.) and the solution filtered to isolate the product. This yielded B6 (45.4 g, 80%) as a white powder. Preparation of B9 (Methyl 3-0-benzyl-2,4-0-dibenzoyl-L-iduronate): The literature cited above provides a multistep process for an analogue of B9. Unexpectedly we have found a superior and more efficient one pot process based on the careful and specific selection of protecting groups. To B6 (2.96 g, 10.4 mmol) was added CH ₃ CN (30 ml_), Me-imidazole (0.92 ml_, 11.4 mmol) and stirred in an ice-bath for 10 min. while kept under argon. Then was added toluenesulfonyl chloride (2.08 g, 10.9 mmol) and stirred 1 hr. followed by addition of another portion of Me-imidazole (0.84 ml_, 10.4 mmol). After another 1 hr. was added another portion of Me-imidazole (1.9 ml_, 23.9 mmol) and benzoyl chloride (2.7 ml_, 22.9 mmol). The cooling was removed and the mixture stirred 2 hrs. at room temperature.

Then was added methanol (15 ml.) and Me-imidazole (0.84 ml_, 10.4 mmol) and stirred another 2 hrs at 50°C. After evaporation the crude was extracted with DCM (100 ml.) and water (100 ml_). The organic phase was dried (MgS0 ₄ ), filtered and evaporated to give a syrup. This syrup was purified by flash column chromatography using EtOAc/hexane (1 :2). This yielded B9-i (3.48 g, 66%) as a white foam.

Preparation of B10 (Methyl 3-0-benzyl-2.4-0-dibenzoyl-1-Q-trichloroacetimidate-L- iduronate):

To B9 (654 mg, 1.29 mmol) was added dry dichloromethane (10 ml_), trichloroacetonitrile (0.65 ml_, 6.45 mmol) and DBU (4 drops) while kept under argon.

The reaction was stirred 45 min., evaporated and immediately purified by flash column chromatography using EtOAc/hexane (1 :4 + 0.1 %NEt ₃ ). This yielded B10 (a/b mixture, 796 mg, 95%) as a white solid. The product may also be crystallized.

Preparation of BA1 (Methyl 3-0-benzyl-2,4-0-dibenzoyl-a-L-idopyranosyluronate)- (1®4)-(Methyl 3-0-benzyl-/V-benzyloxycarbonyl-6-0-benzoyl-a-D- glucosaminopyranoside):

To donor B10 (1.49 g, 2.28 mmol) and acceptor A (1.00 g, 1.92 mmol) was added dry dichloromethane (15 ml.) and dry toluene (10 ml.) and kept under argon. The reaction was cooled in an icebath, TMSOTf (60 mI_, 0.33 mmol) added and stirred 20 min. The reaction was quenched by adding NEt ₃ (0.1 ml.) and extracted with DCM (50 ml.) and NaOH (1M, 50 ml_). The organic phase was separated, dried (MgS0 ₄ ), filtered and evaporated to give a syrup, which was purified by flash column chromatography using toluene/ acetone (20:1 to 10:1). This yielded BA1 (1.78 g, 92%) as a white foam. ES MS: m/z: calcd for [M+Na+]: 1032.3411 ; found: 1032.3356. Preparation of BA2 (Methyl 3-0-benzyl-a-L-idopyranosyluronate)-(1®4)-(Methyl 3-0- benzyl-/V-benzyloxycarbonyl-a-D-qlucosaminopyranoside ^' ):

To BA1 (3.52 g, 3.49 mmol) was added dry MeOH (40 ml.) and NaOMe (5.4 M in MeOH, 0.4 ml.) while kept under argon. The reaction was stirred 2 hrs. and quenched by adding Amberlite IR-120 H+ resin (1.2 g) and stirring another 15 min. The reaction was filtered, evaporated and purified by flash column chromatography using DCM/ MeOH (20:1). This yielded BA2 (2.14 g, 88%) as a white solid. ES MS: m/z: calcd for [M+Na+]: 720.2624; found: 720.2609. Preparation of BA3 (Methyl 3-0-benzyl-2-0-benzoyl-a-L-idopyranosyluronate)-(1®4)-

(Methyl 3-0-benzyl-/V-benzyloxycarbonyl-6-0-benzoyl-a-D-qlucosaminop yranoside):

To BA2 (1.88 g, 2.69 mmol) was added dry dichloromethane (20 ml.) and cooled to - 18°C while kept under argon. To the solution was then added NEt ₃ (1.1 ml_, 8.07 mmol) and BzCI (0.75 ml_, 6.46 mmol). The reaction was stirred 24 hrs, quenched with MeOH (0.2 ml.) and extracted with DCM (100 ml.) and H ₂ 0 (50 ml_). The organic phase was separated, dried (MgS0 ₄ ), filtered and evaporated to give a syrup, which was purified by flash column chromatography using EtOAc/ hexane (1 :2). This yielded BA3 (2.03 g, 83%) as a white foam. The product could also be purified by crystallization from EtOH. ES MS: m/z: calcd for [M+Na+]: 928.3148; found: 928.3103.