FONDAPARINUX

Fondaparinux is a drug belonging to the group of the antithrombotic agents and are used to prevent deep vein thrombosis in patients undergoing orthopedic surgery. It is also used for the treatment of severe venous thrombosis and pulmonary

EMA:Link, US FDA:link

114870-03-0  ………..10x SODIUM SALT

GSK-576428  Org-31540  SR-90107SR-90107A  

launched 2002

Arixtra, Quixidar, Fondaparinux sodium, Fondaparin sodium, Arixtra (TN), Fondaparinux, Org-31540, AC1LCS4W, SR-90107A

Fondaparinux (Arixtra) is a synthetic pentasaccharide anticoagulant. Apart from the O-methyl group at the reducing end of the molecule, the identity and sequence of the five monomeric sugar units contained in fondaparinux is identical to a sequence of five monomeric sugar units that can be isolated after either chemical or enzymatic cleavage of the polymeric glycosaminoglycan heparin and heparan sulfate (HS). This monomeric sequence in heparin and HS is thought to form the high affinity binding site for the natural anti-coagulant factor, antithrombin III (ATIII).

Binding of heparin/HS to ATIII has been shown to increase the anti-coagulant activity of antithrombin III 1000-fold. Fondaparinux potentiates the neutralizing action ofATIII on activated Factor X 300-fold. Fondaparinux may be used: to prevent venous thromboembolism in patients who have undergone orthopedic surgery of the lower limbs (e.g. hip fracture, hip replacement and knee surgery); to prevent VTE in patients undergoing abdominal surgery who are are at high risk of thromboembolic complications; in the treatment of deep vein thrombosis (DVT) and pumonary embolism (PE); in the management of unstable angina (UA) and non-ST segment elevation myocardial infarction (NSTEMI); and in the management of ST segment elevation myocardial infarction (STEMI).

FONDAPARINUX

Fondaparinux (trade name Arixtra) is an anticoagulant medication chemically related to low molecular weight heparins. It is marketed byGlaxoSmithKline. A generic version developed by Alchemia is marketed within the US by Dr. Reddy’s Laboratories.

Fondaparinux is a synthetic pentasaccharide Factor Xa inhibitor. Apart from the O-methyl group at the reducing end of the molecule, the identity and sequence of the five monomeric sugar units contained in fondaparinux is identical to a sequence of five monomeric sugar units that can be isolated after either chemical or enzymatic cleavage of the polymeric glycosaminoglycans heparin and heparan sulfate (HS). Within heparin and heparan sulfate this monomeric sequence is thought to form the high affinity binding site for the anti-coagulant factor antithrombin III (ATIII). Binding of heparin/HS to ATIII has been shown to increase the anti-coagulant activity of antithrombin III 1000 fold. In contrast to heparin, fondaparinux does not inhibit thrombin.

Fondaparinux is given subcutaneously daily. Clinically, it is used for the prevention of deep vein thrombosis in patients who have had orthopedic surgery as well as for the treatment of deep vein thrombosis and pulmonary embolism.

One potential advantage of fondaparinux over LMWH or unfractionated heparin is that the risk for heparin-induced thrombocytopenia (HIT) is substantially lower. Furthermore, there have been case reports of fondaparinux being used to anticoagulate patients with established HIT as it has no affinity to PF-4. However, its renal excretion precludes its use in patients with renal dysfunction.

Unlike direct factor Xa inhibitors, it mediates its effects indirectly through antithrombin III, but unlike heparin, it is selective for factor Xa.^[1]

Fondaparinux is similar to enoxaparin in reducing the risk of ischemic events at nine days, but it substantially reduces major bleeding and improves long term mortality and morbidity.^[2]

It has been investigated for use in conjunction with streptokinase.^[3]

Fondaparinux sodium, a selective coagulation factor Xa inhibitor, was first launched in the U.S. in 2002 by GlaxoSmithKline in a subcutaneous injection formulation for the prophylaxis of deep venous thrombosis (DVT) which may lead to pulmonary embolism in patients at risk for thromboembolic complications who are undergoing hip replacement, knee replacement, hip fracture surgery or abdominal surgery. The product is available in Japan for the treatment of acute deep venous thrombosis and acute pulmonary thromboembolism. In 2004, GlaxoSmithKline launched fondaparinux as an injection to be used in conjunction with warfarin sodium for the subcutaneous treatment of acute pulmonary embolism and DVT.

In 2007, GlaxoSmithKline received approval in the E.U. for the treatment of acute coronary syndrome (ACS), specifically unstable angina or non-ST segment elevation myocardial infarction (UA/NSTEMI) and ST-segment elevation myocardial infarction (STEMI), while in the U.S. an approvable letter was received for this indication. Currently, the drug is in clinical development at GlaxoSmithKline for the treatment of venous limb superficial thrombosis.

Fondaparinux Molecule

GlaxoSmithKline had filed a regulatory application in the E.U. seeking approval of fondaparinux sodium for the prevention of venous thromboembolic events (VTE), however; in 2008, the application was withdrawn for commercial reasons. Commercial launch in Japan for the product for the prevention of venous thromboembolism in high risk patients undergoing surgery in the abdomen took place in 2008.

In 2010, the EMA approved a regulatory application filed by GlaxoSmithKline seeking approval of a once-daily formulation of fondaparinux sodium for the treatment of adults with acute symptomatic spontaneous superficial-vein thrombosis (SVT) of the lower limbs without concomitant DVT. Product launch took place in the U.K. for this indication the same year.

The antithrombotic activity of fondaparinux is the result of antithrombin III (ATIII)-mediated selective inhibition of Factor Xa. By selectively binding to ATIII, the drug potentiates (about 300 times) the innate neutralization of Factor Xa by ATIII. Neutralization of Factor Xa, in turn, interrupts the blood coagulation cascade and thus inhibits thrombin formation and thrombus development. Fondaparinux does not inactivate thrombin (activated Factor II) and has no known effect on platelet function. At the recommended dose, no effects have been demonstrated on fibrinolytic activity or bleeding time.

Originally developed by Organon and Sanofi (formerly known as sanofi-aventis), fondaparinux sodium is currently available in approximately 30 countries. In 2004, Organon transferred its rights to the drug to Sanofi in exchange for revenues based on future sales from jointly developed antithrombotic products and in early 2005, GlaxoSmithKline also acquired the antithrombotic.

At the beginning of 2005, GlaxoSmithKline signed a two-year agreement with Adolor (acquired by Cubist in 2011) for the copromotion of fondaparinux sodium in the U.S. In Sepetember 2013, Aspen Pharmacare acquired Arixtra global rights (excluding China, India and Pakistan) from GlaxoSmithKline for the treatment of thrombosis with GlaxoSmithKline commercializing the product in Indonesia under licence from Aspen.

Chemical structure

Abbreviations

• GlcNS6S = 2-deoxy-6-O-sulfo-2-(sulfoamino)-α-D-glucopyranoside
• GlcA = β-D-glucopyranuronoside
• GlcNS3,6S = 2-deoxy-3,6-di-O-sulfo-2-(sulfoamino)-α-D-glucopyranosyl
• IdoA2S = 2-O-sulfo-α-L-idopyranuronoside
• GlcNS6SOMe = methyl-O-2-deoxy-6-O-sulfo-2-(sulfoamino)-α-D-glucopyranoside

The sequence of monosaccharides is D-GlcNS6S-α-(1,4)-D-GlcA-β-(1,4)-D-GlcNS3,6S-α-(1,4)-L-IdoA2S-α-(1,4)-D-GlcNS6S-OMe, as shown in the following structure:

ARIXTRA (fondaparinux sodium) Injection is a sterile solution containing fondaparinux sodium. It is a synthetic and specific inhibitor of activatedFactor X (Xa). Fondaparinux sodium is methyl O-2-deoxy-6-O-sulfo-2-(sulfoamino)-α-D-glucopyranosyl-(1→4)-O-β-D-glucopyranuronosyl-( 1→4)-O-2-deoxy-3,6-di-O-sulfo-2-(sulfoamino)-α-D-glucopyranosyl-(1→4)-O-2-Osulfo-α-L-idopyranuronosyl-(1→4)-2-deoxy-6-O-sulfo-2-(sulfoamino)-α-D-glucopyranoside, decasodium salt.

The molecular formula of fondaparinux sodium is C₃₁H₄₃N₃Na₁₀O₄₉S₈ and its molecular weight is 1728. The structural formula is provided below:

ARIXTRA is supplied as a sterile, preservative-free injectable solution for subcutaneous use.

Each single-dose, prefilled syringe of ARIXTRA, affixed with an automatic needle protection system, contains 2.5 mg of fondaparinux sodium in 0.5 mL, 5.0 mg of fondaparinux sodium in 0.4 mL, 7.5 mg of fondaparinux sodium in 0.6 mL, or 10.0 mg of fondaparinux sodium in 0.8 mL of an isotonic solutionof sodium chloride and water for injection. The final drug product is a clear and colorless to slightly yellow liquid with a pH between 5.0 and 8.0.

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INTRODUCTION

In U.S. Patent No. 7,468,358, Fondaparinux sodium is described as the “only anticoagulant thought to be completely free of risk from HIT-2 induction.” The biochemical and pharmacologic rationale for the development of a heparin pentasaccharide in Thromb. Res., 86(1), 1-36, 1997 by Walenga et al. cited the recently approved synthetic pentasaccharide Factor Xa inhibitor Fondaparinux sodium. Fondaparinux has also been described in Walenga et al., Expert Opin. Investig. Drugs, Vol. 11, 397-407, 2002 and Bauer, Best Practice & Research Clinical Hematology, Vol. 17, No. 1, 89-104, 2004.

Fondaparinux sodium is a linear octasulfated pentasaccharide (oligosaccharide with five monosaccharide units ) molecule having five sulfate esters on oxygen (O-sulfated moieties) and three sulfates on a nitrogen (N- sulfated moieties). In addition, Fondaparinux contains five hydroxyl groups in the molecule that are not sulfated and two sodium carboxylates. Out of five saccharides, there are three glucosamine derivatives and one glucuronic and one L-iduronic acid. The five saccharides are connected to each other in alternate α and β glycosylated linkages (see Figure 1).

Figure 1 Fondaparinux Sodium

Monosaccharide E Monosaccharide D Monosaccharide C Monosaccharide B Monosaccharide A derived from derived from derived from derived from derived from

Monomer E Monomer D Monomer C Monomer B1 Monomer A2

Fondaparinux Sodium

Fondaparinux sodium is a chemically synthesized methoxy derivative of the natural pentasaccharide sequence, which is the active site of heparin that mediates the interaction with antithrombin (Casu et al., J. Biochem., 197, 59, 1981). It has a challenging pattern of O- and N- sulfates, specific glycosidic stereochemistry, and repeating units of glucosamines and uronic acids (Petitou et al, Progress in the Chemistry of Organic Natural Product, 60, 144-209, 1992).

The monosaccharide units comprising the Fondaparinux molecule are labeled as per the convention in Figure 1, with the glucosamine unit on the right referred to as monosaccharide A and the next, an uronic acid unit to its left as B and subsequent units, C, D and E respectively. The chemical synthesis of Fondaparinux starts with monosaccharides of defined structures that are themselves referred to as Monomers A2, Bl, C, D and E, for differentiation and convenience, and they become the corresponding monosaccharides in fondaparinux sodium.

Due to this complex mixture of free and sulfated hydroxyl groups, and the presence of N- sulfated moieties, the design of a synthetic route to Fondaparinux requires a careful strategy of protection and de-protection of reactive functional groups during synthesis of the molecule. Previously described syntheses of Fondaparinux all adopted a similar strategy to complete the synthesis of this molecule. This strategy can be envisioned as having four stages.

The strategy in the first stage requires selective de-protection of five out of ten hydroxyl groups. During the second stage these five hydroxyls are selectively sulfonated. The third stage of the process involves the de -protection of the remaining five hydroxyl groups. The fourth stage of the process is the selective sulfonation of the 3 amino groups, in the presence of five hydroxyl groups that are not sulfated in the final molecule. This strategy can be envisioned from the following fully protected pentasaccharide, also referred to as the late-stage intermediate.

In this strategy, all of the hydroxyl groups that are to be sulfated are protected with an acyl protective group, for example, as acetates (R = CH₃) or benzoates (R = aryl) (Stages 1 and 2) All of the hydroxyl groups that are to remain as such are protected with benzyl group as benzyl ethers (Stage 3). The amino group, which is subsequently sulfonated, is masked as an azide (N₃) moiety (Stage 4). R¹ and R² are typically sodium in the active pharmaceutical compound (e.g., Fondaparinux sodium).

This strategy allows the final product to be prepared by following the synthetic operations as outlined below: a) Treatment of the late- stage intermediate with base to hydrolyze (deprotect) the acyl ester groups to reveal the five hydroxyl groups. The two R¹ and R² ester groups are hydrolyzed in this step as well.

b) Sulfonation of the newly revealed hydroxyl groups.

c) Hydrogenation of the O-sulfated pentasaccharide to de-benzylate the five benzyl- protected hydroxyls, and at the same time, unmask the three azides to the corresponding amino groups.

d) On the last step of the operation, the amino groups are sulfated selectively at a high pH, in the presence of the five free hydroxyls to give Fondaparinux (Figure 1). While the above strategy has been shown to be viable, it is not without major drawbacks. One drawback lies in the procedure leading to the fully protected pentasaccharide (late stage intermediate), especially during the coupling of the D-glucuronic acid to the next adjacent glucose ring (the D-monomer to C-monomer in the EDCBA nomenclature shown in Figure 1). Sugar oligomers or oligosaccharides, such as Fondaparinux, are assembled using coupling reactions, also known as glycosylation reactions, to “link” sugar monomers together. The difficulty of this linking step arises because of the required stereochemical relationship between the D-sugar and the C-sugar, as shown below:

The stereochemical arrangement illustrated above in Figure 2 is described as having a β- configuration at the anomeric carbon of the D-sugar (denoted by the arrow). The linkage between the D and C units in Fondaparinux has this specific stereochemistry. There are, however, competing β- and α-glycosylation reactions.

The difficulties of the glycosylation reaction in the synthesis of Fondaparinux is well known. In 1991 Sanofi reported a preparation of a disaccharide intermediate in 51% yield having a 12/1 ratio of β/α stereochemistry at the anomeric position (Duchaussoy et al., Bioorg. & Med. Chem. Lett., 1(2), 99-102, 1991).

In another publication (Sinay et al, Carbohydrate Research, 132, C5-C9, 1984) yields on the order of 50% with coupling times on the order of 6- days are reported. U.S. Patent No. 4,818,816 {see e.g., column 31, lines 50-56) discloses a 50% yield for the β-glycosylation.

Alchemia’s U.S. Patent No. 7,541,445 is even less specific as to the details of the synthesis of this late-stage Fondaparinux synthetic intermediate. The ‘445 Patent discloses several strategies for the assembly of the pentasaccharide (1+4, 3+2 or 2+3) using a 2-acylated D-sugar (specifically 2-allyloxycarbonyl) for the glycosylation coupling reactions. However, Alchemia’s strategy involves late-stage pentasaccharides that all incorporate a 2-benzylated D- sugar.

The transformation of acyl to benzyl is performed either under acidic or basic conditions. Furthermore, these transformations, using benzyl bromide or benzyl trichloroacetimidate, typically result in extensive decomposition and the procedure suffers from poor yields. Thus, such transformations (at a disaccharide, trisaccharide, and pentasaccharide level) are typically not acceptable for industrial scale production.

Examples of fully protected pentasaccharides are described in Duchaussoy et al, Bioorg. Med. Chem. Lett., 1 (2), 99-102, 1991; Petitou et al, Carbohydr. Res., 167, 67-75, 1987; Sinay et al, Carbohydr. Res., 132, C5-C9, 1984; Petitou et al., Carbohydr. Res., 1147, 221-236, 1986; Lei et al., Bioorg. Med. Chem., 6, 1337-1346, 1998; Ichikawa et al., Tet. Lett., 27(5), 611-614, 1986; Kovensky et al, Bioorg. Med. Chem., 1999, 7, 1567-1580, 1999.

These fully protected pentasaccharides may be converted to the O- and N-sulfated pentasaccharides using the four steps (described earlier) of: a) saponification with LiOHZH₂CVNaOH, b) O-sulfation by an Et₃N- SO₃ complex; c) de-benzylation and azide reduction via H₂/Pd hydrogenation; and d) N-sulfation with a pyridine-SO₃ complex.

Even though many diverse analogs of the fully protected pentasaccharide have been prepared, none use any protective group at the 2-position of the D unit other than a benzyl group. Furthermore, none of the fully protected pentasaccharide analogs offer a practical, scaleable and economical method for re-introduction of the benzyl moiety at the 2-position of the D unit after removal of any participating group that promotes β-glycosylation.

Furthermore, the coupling of benzyl protected sugars proves to be a sluggish, low yielding and problematic process, typically resulting in substantial decomposition of the pentasaccharide (prepared over 50 synthetic steps), thus making it unsuitable for a large [kilogram] scale production process.

Ref. 1. Sinay et al, Carbohydr. Res., 132, C5-C9, 1984.

Ref. 2. Petitou et al., Carbohydr. Res., 147, 221-236. 1986

It has been a general strategy for carbohydrate chemists to use base-labile ester-protecting group at 2-position of the D unit to build an efficient and stereoselective β-glycosidic linkage. To construct the β-linkage carbohydrate chemists have previously acetate and benzoate ester groups, as described, for example, in the review by Poletti et al., Eur. J. Chem., 2999-3024, 2003.

The ester group at the 2-position of D needs to be differentiated from the acetate and benzoates at other positions in the pentasaccharide. These ester groups are hydrolyzed and sulfated later in the process and, unlike these ester groups, the 2-hydroxyl group of the D unit needs to remain as the hydroxyl group in the final product, Fondaparinux sodium.

Some of the current ester choices for the synthetic chemists in the field include methyl chloro acetyl and chloro methyl acetate [MCA or CMA] . The mild procedures for the selective removal of theses groups in the presence of acetates and benzoates makes them ideal candidates. However, MCA/CMA groups have been shown to produce unwanted and serious side products during the glycosylation and therefore have not been favored in the synthesis of Fondaparinux sodium and its analogs. For by-product formation observed in acetate derivatives see Seeberger et al., J. Org. Chem., 2004, 69, 4081-93.

Similar by-product formation is also observed using chloroacetate derivatives. See Orgueira et al., Eur. J. Chem., 9(1), 140-169, 2003.

The levulinyl group can be rapidly and almost quantitatively removed by treatment with hydrazine hydrate as the deprotection reagent as illustrated in the example below. Under the same reaction conditions primary and secondary acetate and benzoate esters are hardly affected by hydrazine hydrate. See, e.g., Seeberger et al, J. Org. Chem., 69, 4081-4093, 2004.

The syntheses of Fondaparinux sodium described herein takes advantage of the levulinyl group in efficient construction of the trisaccharide EDC with improved β- selectivity for the coupling under milder conditions and increased yields.

Substitution of the benzyl protecting group with a THP moiety provides its enhanced ability to be incorporated quantitatively in position-2 of the unit D of the pentasaccharide. Also, the THP group behaves in a similar manner to a benzyl ether in terms of function and stability. In the processes described herein, the THP group is incorporated at the 2-position of the D unit at this late stage of the synthesis (i.e., after the D and C units have been coupled through a 1,2-trans glycosidic (β-) linkage). The THP protective group typically does not promote an efficient β- glycosylation and therefore is preferably incorporated in the molecule after the construction of the β-linkage.

Fondaparinux and sodium salt thereof can be prepared from pure compound of Formula II by following the teachings from Bioorganic and Medicinal Chemistry Letters, 1(2), p. 95-98 (1991). A second aspect of the present invention provides a process for the preparation of 4-0- -D-glucopyranosyl-l,6-anhydro- -D-glucopyranose, represented by STR BELOW

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SYNTHESIS

EP2464668A2   AND US8288515

The scheme below exemplifies some of the processes of the present invention disclosed herein.

The tetrahydropyranyl (THP) protective group and the benzyl ether protective group are suitable hydroxyl protective groups and can survive the last four synthetic steps (described above) in the synthesis of Fondaparinux sodium, even under harsh reaction conditions. Certain other protecting groups do not survive the last four synthetic steps in high yield.

Synthesis of Fondaparinux

Fondaparinux was prepared using the following procedure:

Synthesis of Fondaparinux

Fondaparinux was prepared using the following procedure:

The ester moieties in EDCBA Pentamer were hydrolyzed with sodium and lithium hydroxide in the presence of hydrogen peroxide in dioxane mixing at room temperature for 16 hours to give the pentasaccharide intermediate API1. The five hydroxyl moieties in API1 were sulfated using a pyridine-sulfur trioxide complex in dimethylformamide, mixing at 60° C. for 2 hours and then purified using column chromatography (CG-161), to give the pentasulfated pentasaccharide API2. The intermediate API2 was then hydrogenated to reduce the three azides on sugars E, C and A to amines and the reductive deprotection of the five benzyl ethers to their corresponding hydroxyl groups to form the intermediate API3. This transformation occurs by reacting API2 with 10% palladium/carbon catalyst with hydrogen gas for 72 hours. The three amines on API3 were then sulfated using the pyridine-sulfur trioxide complex in sodium hydroxide and ammonium acetate, allowing the reaction to proceed for 12 hours. The acidic work-up procedure of the reaction removes the THP group to provide crude fondaparinux which is purified and is subsequently converted to its salt form. The crude mixture was purified using an ion-exchange chromatographic column (HiQ resin) followed by desalting using a size exclusion resin or gel filtration (Biorad Sephadex G25) to give the final API, fondaparinux sodium

Experimental Procedures Preparation of IntD1 Bromination of Glucose Pentaacetate

To a 500 ml flask was added 50 g of glucose pentaacetate (C₆H₂₂O₁₁) and 80 ml of methylene chloride. The mixture was stirred at ice-water bath for 20 min HBr in HOAc (33%, 50 ml) was added to the reaction mixture. After stirring for 2.5 hr another 5 ml of HBr was added to the mixture. After another 30 min, the mixture was added 600 ml of methylene chloride. The organic mixture was washed with cold water (200 ml×2), Saturated NaHCO₃(200 ml×2), water (200 ml) and brine (200 ml×2). The organic layer was dried over Na₂SO₄ and the mixture was evaporated at RT to give white solid as final product, bromide derivative, IntD1 (˜95% yield). C₁₄H₁₉BrO₉, TLC R_f=0.49, SiO₂, 40% ethyl acetate/60% hexanes; Exact Mass 410.02.

Preparation of IntD2 by Reductive Cyclization

To a stirring mixture of bromide IntD1 (105 g), tetrabutylammonium iodide (60 g, 162 mmol) and activated 3 Å molecular sieves in anhydrous acetonitrile (2 L), solid NaBH₄ (30 g, 793 mmol) was added. The reaction was heated at 40° C. overnight. The mixture was then diluted with dichloromethane (2 L) and filtered through Celite®. After evaporation, the residue was dissolved in 500 ml ethyl acetate. The white solid (Bu₄NI or Bu₄NBr) was filtered. The ethyl acetate solution was evaporated and purified by chromatography on silica gel using ethyl acetate and hexane as eluent to give the acetal-triacetate IntD2 (˜60-70% yield). TLC R_f=0.36, SiO₂ in 40% ethyl acetate/60% hexanes.

Preparation of IntD3 by De-Acetylation

To a 1000 ml flask was added triacetate IntD2 (55 g) and 500 ml of methanol. After stirring 30 min, the reagent NaOMe (2.7 g, 0.3 eq) was added and the reaction was stirred overnight. Additional NaOMe (0.9 g) was added to the reaction mixture and heated to 50° C. for 3 hr. The mixture was neutralized with Dowex 50Wx8 cation resin, filtered and evaporated. The residue was purified by silica gel column to give 24 g of trihydroxy-acetal IntD3. TLC R_f=0.36 in SiO₂, 10% methanol/90% ethyl acetate.

Preparation of IntD4 by Benzylidene Formation

To a 1000 ml flask was added trihydroxy compound IntD3 (76 g) and benzaldehyde dimethyl acetate (73 g, 1.3 eq). The mixture was stirred for 10 min, after which D(+)-camphorsulfonic acid (8.5 g, CSA) was added. The mixture was heated at 50° C. for two hours. The reaction mixture was then transferred to separatory funnel containing ethyl acetate (1.8 L) and sodium bicarbonate solution (600 ml). After separation, the organic layer was washed with a second sodium bicarbonate solution (300 ml) and brine (800 ml). The two sodium carbonate solutions were combined and extracted with ethyl acetate (600 ml×2). The organic mixture was evaporated and purified by silica gel column to give the benzylidene product IntD4 (77 g, 71% yield). TLC R_f=0.47, SiO₂ in 40% ethyl acetate/60% hexanes.

Preparation of IntD5 by Benzylation

To a 500 ml flask was added benzylidene acetal compound IntD4 (21 g,) in 70 ml THF. To another flask (1000 ml) was added NaH (2 eq). The solution of IntD4 was then transferred to the NaH solution at 0° C. The reaction mixture was stirred for 30 min, then benzyl bromide (16.1 ml, 1.9 eq) in 30 ml THF was added. After stirring for 30 min, DMF (90 ml) was added to the reaction mixture. Excess NaH was neutralized by careful addition of acetic acid (8 ml). The mixture was evaporated and purified by silica gel column to give the benzyl derivative IntD5. (23 g) TLC R_f=0.69, SiO₂ in 40% ethyl acetate/60% hexanes.

Preparation of IntD6 by Deprotection of Benzylidene

To a 500 ml flask was added the benzylidene-acetal compound IntD5 (20 g) and 250 ml of dichloromethane, the reaction mixture was cooled to 0° C. using an ice-water-salt bath. Aqueous TFA (80%, 34 ml) was added to the mixture and stirred over night. The mixture was evaporated and purified by silica gel column to give the dihydroxy derivative IntD6. (8 g, 52%). TLC R_f=0.79, SiO₂ in 10% methanol/90% ethyl acetate.

Preparation of IntD7 by Oxidation of 6-Hydroxyl

To a 5 L flask was added dihydroxy compound IntD6 (60 g), TEMPO (1.08 g), sodium bromide (4.2 g), tetrabutylammonium chloride (5.35 g), saturated NaHCO₃ (794 ml) and EtOAc (1338 ml). The mixture was stirred over an ice-water bath for 30 min To another flask was added a solution of NaOCl (677 ml), saturated NaHCO₃ (485 ml) and brine (794 ml). The second mixture was added slowly to the first mixture (over about two hrs). The resulting mixture was then stirred overnight. The mixture was separated, and the inorganic layer was extracted with EtOAc (800 ml×2). The combined organic layers were washed with brine (800 ml). Evaporation of the organic layer gave 64 g crude carboxylic acid product IntD7 which was used in the next step use without purification. TLC R_f=0.04, SiO₂ in 10% methanol/90% ethyl acetate.

Preparation of Monomer D by Benzylation of the Carboxylic Acid

To a solution of carboxylic acid derivative IntD7 (64 g) in 600 ml of dichloromethane, was added benzyl alcohol (30 g) and N-hydroxybenzotriazole (80 g, HOBt). After stirring for 10 min triethylamine (60.2 g) was added slowly. After stirring another 10 min, dicyclohexylcarbodiimide, (60.8 g, DCC) was added slowly and the mixture was stirred overnight. The reaction mixture was filtered and the solvent was removed under reduced pressure followed by chromatography on silica gel to provide 60.8 g (75%, over two steps) of product, Monomer D. TLC R_f=0.51, SiO₂ in 40% ethyl acetate/60% hexanes.

Synthesis of the BA Dimer

Step 1. Preparation of BMod1, Levulination of Monomer B1

A 100 L reactor was charged with 7.207 Kg of Monomer B1 (21.3 moles, 1 equiv), 20 L of dry tetrahydrofuran (THF) and agitated to dissolve. When clear, it was purged with nitrogen and 260 g of dimethylamino pyridine (DMAP, 2.13 moles, 0.1 equiv) and 11.05 L of diisopropylethylamine (DIPEA, 8.275 kg, 63.9 moles, 3 equiv) was charged into the reactor. The reactor was chilled to 10-15° C. and 13.7 kg levulinic anhydride (63.9 mol, 3 equiv) was transferred into the reactor. When the addition was complete, the reaction was warmed to ambient temperature and stirred overnight or 12-16 hours. Completeness of the reaction was monitored by TLC (40:60 ethyl acetate/hexane) and HPLC. When the reaction was complete, 20 L of 10% citric acid, 10 L of water and 25 L of ethyl acetate were transferred into the reactor. The mixture was stirred for 30 min and the layers were separated. The organic layer (EtOAc layer) was extracted with 20 L of water, 20 L 5% sodium bicarbonate and 20 L 25% brine solutions. The ethyl acetate solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.) and dried overnight. The yield of the isolated syrup of BMod1 was 100%.

Synthesis of the BA Dimer

Step 2. Preparation of BMod2, TFA Hydrolysis of BMod1

A 100 L reactor was charged with 9296 Kg of 4-Lev Monomer B1 (BMod1) (21.3 mol, 1 equiv). The reactor chiller was turned to <5° C. and stirring was begun, after which 17.6 L of 90% TFA solution (TFA, 213 mole, 10 equiv) was transferred into the reactor. When the addition was complete, the reaction was monitored by TLC and HPLC. The reaction took approximately 2-3 hours to reach completion. When the reaction was complete, the reactor was chilled and 26.72 L of triethylamine (TEA, 19.4 Kg, 191.7 mole, 0.9 equiv) was transferred into the reactor. An additional 20 L of water and 20 L ethyl acetate were transferred into the reactor. This was stirred for 30 min and the layers were separated. The organic layer was extracted (EtOAc layer) with 20 L 5% sodium bicarbonate and 20 L 25% brine solutions. The ethyl acetate solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The crude product was purified in a 200 L silica column using 140-200 L each of the following gradient profiles: 50:50, 80:20 (EtOAc/heptane), 100% EtOAc, 5:95, 10:90 (MeOH/EtOAc). The pure fractions were pooled and evaporated to a syrup. The yield of the isolated syrup, BMod2 was 90%.

Synthesis of the BA Dimer

Step 3. Preparation of BMod3, Silylation of BMod2

A 100 L reactor was charged with 6.755 Kg 4-Lev-1,2-DiOH Monomer B1 (BMod2) (17.04 mol, 1 equiv), 2328 g of imidazole (34.2 mol, 2 equiv) and 30 L of dichloromethane. The reactor was purged with nitrogen and chilled to −20° C., then 5.22 L tert-butyldiphenylchloro-silane (TBDPS-Cl, 5.607 Kg, 20.4 mol, 1.2 equiv) was transferred into the reactor. When addition was complete, the chiller was turned off and the reaction was allowed to warm to ambient temperature. The reaction was monitored by TLC (40% ethyl acetate/hexane) and HPLC. The reaction took approximately 3 hours to reach completion. When the reaction was complete, 20 L of water and 10 L of DCM were transferred into the reactor and stirred for 30 min, after which the layers were separated. The organic layer (DCM layer) was extracted with 20 L water and 20 L 25% brine solutions. Dichloromethane solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The yield of BMod3 was about 80%.

Synthesis of the BA Dimer

Step 4. Preparation of BMod4, Benzoylation

A 100 L reactor was charged with 8.113 Kg of 4-Lev-1-Si-2-OH Monomer B1 (BMod3) (12.78 mol, 1 equiv), 9 L of pyridine and 30 L of dichloromethane. The reactor was purged with nitrogen and chilled to −20° C., after which 1.78 L of benzoyl chloride (2155 g, 15.34 mol, 1.2 equiv) was transferred into the reactor. When addition was complete, the reaction was allowed to warm to ambient temperature. The reaction was monitored by TLC (40% ethyl acetate/heptane) and HPLC. The reaction took approximately 3 hours to reach completion. When the reaction was complete, 20 L of water and 10 L of DCM were transferred into the reactor and stirred for 30 min, after which the layers were separated. The organic layer (DCM layer) was extracted with 20 L water and 20 L 25% brine solutions. The DCM solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). Isolated syrup BMod4 was obtained in 91% yield.

Synthesis of the BA Dimer

Step 5. Preparation of BMod5, Desilylation

A 100 L reactor was charged with 8.601 Kg of 4-Lev-1-Si-2-Bz Monomer B1 (BMod4) (11.64 mol, 1 equiv) in 30 L terahydrofuran. The reactor was purged with nitrogen and chilled to 0° C., after which 5.49 Kg of tetrabutylammonium fluoride (TBAF, 17.4 mol, 1.5 equiv) and 996 mL (1045 g, 17.4 mol, 1.5 equiv) of glacial acetic acid were transferred into the reactor. When the addition was complete, the reaction was stirred at ambient temperature. The reaction was monitored by TLC (40:60 ethyl acetate/hexane) and HPLC. The reaction took approximately 6 hours to reach completion. When the reaction was complete, 20 L of water and 10 L of DCM were transferred into the reactor and stirred for 30 min, after which the layers were separated. The organic layer (DCM layer) was extracted with 20 L water and 20 L 25% brine solutions. The dichloromethane solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The crude product was purified in a 200 L silica column using 140-200 L each of the following gradient profiles: 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 (EtOAc/heptane) and 200 L 100% EtOAc. Pure fractions were pooled and evaporated to a syrup. The intermediate BMod5 was isolated as a syrup in 91% yield.

Synthesis of the BA Dimer

Step 6: Preparation of BMod6, TCA Formation

A 100 L reactor was charged with 5.238 Kg of 4-Lev-1-OH-2-Bz Monomer B1 (BMod5) (10.44 mol, 1 equiv) in 30 L of DCM. The reactor was purged with nitrogen and chilled to 10-15° C., after which 780 mL of diazabicyclo undecene (DBU, 795 g, 5.22 mol, 0.5 equiv) and 10.47 L of trichloroacetonitrile (TCA, 15.08 Kg, 104.4 mol, 10 equiv) were transferred into the reactor. Stirring was continued and the reaction was kept under a nitrogen atmosphere. After reagent addition, the reaction was allowed to warm to ambient temperature. The reaction was monitored by HPLC and TLC (40:60 ethyl acetate/heptane). The reaction took approximately 2 hours to reach completion. When the reaction was complete, 20 L of water and 10 L of dichloromethane were transferred into the reactor. This was stirred for 30 min and the layers were separated. The organic layer (DCM layer) was separated with 20 L water and 20 L 25% brine solutions. The dichloromethane solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The crude product was purified in a 200 L silica column using 140-200 L each of the following gradient profiles: 10:90, 20:80, 30:70, 40:60 and 50:50 (EtOAc/Heptane). Pure fractions were pooled and evaporated to a syrup. The isolated yield of BMod6 was 73%.

Synthesis of the BA Dimer

Step 7. Preparation of AMod1, Acetylation of Monomer A2

A 100 L reactor was charged with 6.772 Kg of Monomer A2 (17.04 mole, 1 eq.), 32.2 L (34.8 Kg, 340.8 moles, 20 eq.) of acetic anhydride and 32 L of dichloromethane. The reactor was purged with nitrogen and chilled to −20° C. When the temperature reached −20° C., 3.24 L (3.63 Kg, 25.68 mol, 1.5 equiv) of boron trifluoride etherate (BF₃.Et₂O) was transferred into the reactor. After complete addition of boron trifluoride etherate, the reaction was allowed to warm to room temperature. The completeness of the reaction was monitored by HPLC and TLC (30:70 ethyl acetate/heptane). The reaction took approximately 3-5 hours for completion. When the reaction was complete, extraction was performed with 3×15 L of 10% sodium bicarbonate and 20 L of water. The organic phase (DCM) was evaporated to a syrup (bath temp. 40° C.) and allowed to dry overnight. The syrup was purified in a 200 L silica column using 140 L each of the following gradient profiles: 5:95, 10:90, 20:80, 30:70, 40:60 and 50:50 (EtOAc/heptane). Pure fractions were pooled and evaporated to a syrup. The isolated yield of AMod1 was 83%.

Synthesis of the BA Dimer

Step 8. Preparation of AMod3,1-Methylation of AMod1

A 100 L reactor was charged with 5891 g of acetyl Monomer A2 (AMod1) (13.98 mole, 1 eq.) in 32 L of dichloromethane. The reactor was purged with nitrogen and was chilled to 0° C., after which 2598 mL of trimethylsilyl iodide (TMSI, 3636 g, 18 mol, 1.3 equiv) was transferred into the reactor. When addition was complete, the reaction was allowed to warm to room temperature. The completeness of the reaction was monitored by HPLC and TLC (30:70 ethyl acetate/heptane). The reaction took approximately 2-4 hours to reach completion. When the reaction was complete, the mixture was diluted with 20 L of toluene. The solution was evaporated to a syrup and was co-evaporated with 3×6 L of toluene. The reactor was charged with 36 L of dichloromethane (DCM), 3.2 Kg of dry 4 Å Molecular Sieves, 15505 g (42 mol, 3 equiv) of tetrabutyl ammonium iodide (TBAI) and 9 L of dry methanol. This was stirred until the TBAI was completely dissolved, after which 3630 mL of diisopropyl-ethylamine (DIPEA, 2712 g, 21 moles, 1.5 equiv) was transferred into the reactor in one portion. The completion of the reaction was monitored by HPLC and TLC (30:70 ethyl acetate/heptane). The reaction took approximately 16 hours for completion. When the reaction was complete, the molecular sieves were removed by filtration. Added were 20 L EtOAc and extracted with 4×20 L of 25% sodium thiosulfate and 20 L 10% NaCl solutions. The organic layer was separated and dried with 8-12 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The crude product was purified in a 200 L silica column using 140-200 L each of the following gradient profiles: 5:95, 10:90, 20:80, 30:70 and 40:60 (EtOAc/heptane). The pure fractions were pooled and evaporated to give intermediate AMod3 as a syrup. The isolated yield was 75%.

Synthesis of the BA Dimer

Step 9. Preparation of AMod4, DeAcetylation of AMod3

A 100 L reactor was charged with 4128 g of 1-Methyl 4,6-Diacetyl Monomer A2 (AMod3) (10.5 mol, 1 equiv) and 18 L of dry methanol and dissolved, after which 113.4 g (2.1 mol, 0.2 equiv) of sodium methoxide was transferred into the reactor. The reaction was stirred at room temperature and monitored by TLC (40% ethyl acetate/hexane) and HPLC. The reaction took approximately 2-4 hours for completion. When the reaction was complete, Dowex 50Wx8 cation resin was added in small portions until the pH reached 6-8. The Dowex 50Wx8 resin was filtered and the solution was evaporated to a syrup (bath temp. 40° C.). The syrup was diluted with 10 L of ethyl acetate and extracted with 20 L brine and 20 L water. The ethyl acetate solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.) and dried overnight at the same temperature. The isolated yield of the syrup AMod4 was about 88%.

Synthesis of the BA Dimer

Step 10. Preparation of AMod5,6-Benzoylation

A 100 L reactor was charged with 2858 g of Methyl 4,6-diOH Monomer A2 (AMod4) (9.24 mol, 1 equiv) and co-evaporated with 3×10 L of pyridine. When evaporation was complete, 15 L of dichloromethane, 6 L of pyridine were transferred into the reactor and dissolved. The reactor was purged with nitrogen and chilled to −40° C. The reactor was charged with 1044 mL (1299 g, 9.24 mol, 1 equiv) of benzoyl chloride. When the addition was complete, the reaction was allowed to warm to −10° C. over a period of 2 hours. The reaction was monitored by TLC (60% ethyl acetate/hexane). When the reaction was completed, the solution was evaporated to a syrup (bath temp. 40° C.). This was co-evaporated with 3×15 L of toluene. The syrup was diluted with 40 L ethyl acetate. Extraction was carried out with 20 L of water and 20 L of brine solution. The Ethyl acetate solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The crude product was purified in a 200 L silica column using 140-200 L each of the following gradient profiles: 5:95, 10:90, 20:80, 25:70 and 30:60 (EtOAc/heptane). The pure fractions were pooled and evaporated to a syrup. The isolated yield of the intermediate AMod5 was 84%.

Synthesis of the BA Dimer

Step 11. Preparation of BA1, Coupling of Amod5 with BMod6

A 100 L reactor was charged with 3054 g of methyl 4-Hydroxy-Monomer A2 (AMod5) from Step 10 (7.38 mol, 1 equiv) and 4764 g of 4-Lev-1-TCA-Monomer B1 (BMod6) from Step 6 (7.38 mol, 1 equiv). The combined monomers were dissolved in 20 L of toluene and co-evaporated at 40° C. Co evaporation was repeated with an additional 2×20 L of toluene, after which 30 L of dichloromethane (DCM) was transferred into the reactor and dissolved. The reactor was purged with nitrogen and was chilled to below −20° C. When the temperature was between −20° C. and −40° C., 1572 g (1404 mL, 11.12 moles, 1.5 equiv) of boron trifluoride etherate (BF₃.Et₂O) were transferred into the reactor. After complete addition of boron trifluoride etherate, the reaction was allowed to warm to 0° C. and stirring was continued. The completeness of the reaction was monitored by HPLC and TLC (40:70 ethyl acetate/heptane). The reaction required 3-4 hours to reach completion. When the reaction was complete, 926 mL (672 g, 6.64 mol, 0.9 equiv) of triethylamine (TEA) was transferred into the mixture and stirred for an additional 30 minutes, after which 20 L of water and 10 L of dichloromethane were transferred into the reactor. The solution was stirred for 30 min and the layers were separated. The organic layer (DCM layer) was separated with 2×20 L water and 20 L 25% 4:1 sodium chloride/sodium bicarbonate solution. The dichloromethane solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.) and used in the next step. The isolated yield of the disaccharide BA1 was about 72%.

Synthesis of the BA Dimer

Step 12, Removal of Levulinate Methyl [(methyl 2-O-benzoyl-3-O-benzyl-α-L-Idopyranosyluronate)-(1→4)-2-azido-6-O-benzoyl-3-O-benzyl]-2-deoxy-α-D-glucopyranoside

A 100 L reactor was charged with 4.104 Kg of 4-Lev BA Dimer (BA1) (4.56 mol, 1 equiv) in 20 L of THF. The reactor was purged with nitrogen and chilled to −20 to −25° C., after which 896 mL of hydrazine hydrate (923 g, 18.24 mol, 4 equiv) was transferred into the reactor. Stirring was continued and the reaction was monitored by TLC (40% ethyl acetate/heptane) and HPLC. The reaction took approximately 2-3 hour for the completion, after which 20 L of 10% citric acid, 10 L of water and 25 L of ethyl acetate were transferred into the reactor. This was stirred for 30 min and the layers were separated. The organic layer (ETOAc layer) was extracted with 20 L 25% brine solutions. The ethyl acetate solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The crude product was purified in a 200 L silica column using 140-200 L each of the following gradient profiles: 10:90, 20:80, 30:70, 40:60 and 50:50 (EtOAc/heptane). The pure fractions were pooled and evaporated to dryness. The isolated yield of the BA Dimer was 82%. Formula: C₄₂H₄₃N₃O₁₃; Mol. Wt. 797.80.

Synthesis of the EDC Trimer

Step 1. Preparation of EMod1, Acetylation

A 100 L reactor was charged with 16533 g of Monomer E (45 mole, 1 eq.), 21.25 L acetic anhydride (225 mole, 5 eq.) and 60 L of dichloromethane. The reactor was purged with nitrogen and was chilled to −10° C. When the temperature was at −10° C., 1.14 L (1277 g) of boron trifluoride etherate (BF₃.Et₂O, 9.0 moles, 0.2 eq) were transferred into the reactor. After the complete addition of boron trifluoride etherate, the reaction was allowed to warm to room temperature. The completeness of the reaction was monitored by TLC (30:70 ethyl acetate/heptane) and HPLC. The reaction took approximately 3-6 hours to reach completion. When the reaction was completed, the mixture was extracted with 3×50 L of 10% sodium bicarbonate and SOL of water. The organic phase (DCM) was evaporated to a syrup (bath temp. 40° C.) and allowed to dry overnight. The isolated yield of EMod1 was 97%.

Synthesis of the EDC Trimer

Step 2. Preparation of EMod2, De-Acetylation of Azidoglucose

A 100 L reactor was charged with 21016 g of 1,6-Diacetyl Monomer E (EMod1) (45 mole, 1 eq.), 5434 g of hydrazine acetate (NH₂NH₂.HOAc, 24.75 mole, 0.55 eq.) and 50 L of DMF (dimethyl formamide). The solution was stirred at room temperature and the reaction was monitored by TLC (30% ethyl acetate/hexane) and HPLC. The reaction took approximately 2-4 hours for completion. When the reaction was completed, 50 L of dichloromethane and 40 L of water were transferred into the reactor. This was stirred for 30 minutes and the layers were separated. This was extracted with an additional 40 L of water and the organic phase was dried in 6-8 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.) and dried overnight at the same temperature. The syrup was purified in a 200 L silica column using 140-200 L each of the following gradient profiles: 20:80, 30:70, 40:60 and 50:50 (EtOAc/heptane). Pure fractions were pooled and evaporated to a syrup. The isolated yield of intermediate EMod2 was 100%.

Synthesis of the EDC Trimer

Step 3. Preparation of EMod3, Formation of 1-TCA

A 100 L reactor was charged with 12752 g of 1-Hydroxy Monomer E (EMod2) (30 mole, 1 eq.) in 40 L of dichloromethane. The reactor was purged with nitrogen and stirring was started, after which 2.25 L of DBU (15 moles, 0.5 eq.) and 15.13 L of trichloroacetonitrile (150.9 moles, 5.03 eq) were transferred into the reactor. Stirring was continued and the reaction was kept under nitrogen. After the reagent addition, the reaction was allowed to warm to ambient temperature. The reaction was monitored by TLC (30:70 ethyl acetate/Heptane) and HPLC. The reaction took approximately 2-3 hours to reach completion. When the reaction was complete, 40 L of water and 20 L of DCM were charged into the reactor. This was stirred for 30 min and the layers were separated. The organic layer (DCM layer) was extracted with 40 L water and the DCM solution was dried in 6-8 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The crude product was purified in a 200 L silica column using 140-200 L each of the following gradient profiles: 10:90 (DCM/EtOAc/heptane), 20:5:75 (DCM/EtOAc/heptane) and 20:10:70 DCM/EtOAc/heptane). Pure fractions were pooled and evaporated to give Intermediate EMod3 as a syrup. Isolated yield was 53%.

Synthesis of the EDC Trimer

Step 4. Preparation of ED Dimer, Coupling of E-TCA with Monomer D

A 100 L reactor was charged with 10471 g of 6-Acetyl-1-TCA Monomer E (EMod3) (18.3 mole, 1 eq., FW: 571.8) and 6594 g of Monomer D (16.47 mole, 0.9 eq, FW: 400.4). The combined monomers were dissolved in 20 L toluene and co-evaporated at 40° C. This was repeated with co-evaporation with an additional 2×20 L of toluene, after which 60 L of dichloromethane (DCM) were transferred into the reactor and dissolved. The reactor was purged with nitrogen and was chilled to −40° C. When the temperature was between −30° C. and −40° C., 2423 g (2071 mL, 9.17 moles, 0.5 eq) of TES Triflate were transferred into the reactor. After complete addition of TES Triflate the reaction was allowed to warm and stirring was continued. The completeness of the reaction was monitored by HPLC and TLC (35:65 ethyl acetate/Heptane). The reaction required 2-3 hours to reach completion. When the reaction was completed, 2040 mL of triethylamine (TEA, 1481 g, 0.8 eq.) were transferred into the reactor and stirred for an additional 30 minutes. The organic layer (DCM layer) was extracted with 2×20 L 25% 4:1 sodium chloride/sodium bicarbonate solution. The dichloromethane solution was dried in 6-8 Kg of anhydrous sodium sulfate. The syrup was purified in a 200 L silica column using 140-200 L each of the following gradient profiles: 15:85, 20:80, 25:75, 30:70 and 35:65 (EtOAc/heptane). Pure fractions were pooled and evaporated to a syrup. The ED Dimer was obtained in 81% isolated yield.

Synthesis of the EDC Trimer

Step 5. Preparation of ED1 TFA, Hydrolysis of ED Dimer

A 100 L reactor was charged with 7.5 Kg of ED Dimer (9.26 mol, 1 equiv). The reactor was chilled to <5° C. and 30.66 L of 90% TFA solution (TFA, 370.4 mol, 40 equiv) were transferred into the reactor. When the addition was completed the reaction was allowed to warm to room temperature. The reaction was monitored by TLC (40:60 ethyl acetate/hexanes) and HPLC. The reaction took approximately 3-4 hours to reach completion. When the reaction was completed, was chilled and 51.6 L of triethylamine (TEA, 37.5 Kg, 370.4 mole) were transferred into the reactor, after which 20 L of water & 20 L ethyl acetate were transferred into the reactor. This was stirred for 30 min and the layers were separated. The organic layer (EtOAc layer) was extracted with 20 L 5% sodium bicarbonate and 20 L 25% brine solutions. Ethyl acetate solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The crude product was purified in a 200 L silica column using 140-200 L each of the following gradient profiles: 20:80, 30:70, 40:60, 50:50, 60:40 (EtOAc/heptane). The pure fractions were pooled and evaporated to a syrup. Isolated yield of ED1 was about 70%.

Synthesis of the EDC Trimer

Step 6. Preparation of ED2, Silylation of ED1

A 100 L reactor was charged with 11000 g of 1,2-diOH ED Dimer (ED1) (14.03 mol, 1 equiv), 1910.5 g of imidazole (28.06 mol, 2 equiv) and 30 L of dichloromethane. The reactor was purged with nitrogen and chilled to −20° C., after which 3.53 L butyldiphenylchloro-silane (TBDPS-Cl, 4.628 Kg, 16.835 mol, 1.2 equiv) was charged into the reactor. When the addition was complete, the chiller was turned off and the reaction was allowed to warm to ambient temperature. The reaction was monitored by TLC (50% ethyl acetate/hexane) and HPLC. The reaction required 4-6 hours to reach completion. When the reaction was completed, 20 L of water and 10 L of dichloromethane were transferred into the reactor and stirred for 30 min and the layers were separated. The organic layer (DCM layer) was extracted with 20 L water and 20 L 25% brine solutions. Dichloromethane solution was dried in 4-6 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). Intermediate ED2 was obtained in 75% isolated yield.

Synthesis of the EDC Trimer

Step 7. Preparation of ED3, D-Levulination

A 100 L reactor was charged with 19800 g of 1-Silyl ED Dimer (ED2) (19.37 moles, 1 equiv) and 40 L of dry tetrahydrofuran (THF) and agitated to dissolve. The reactor was purged with nitrogen and 237 g of dimethylaminopyridine (DMAP, 1.937 moles, 0.1 equiv) and 10.05 L of diisopropylethylamine (DIPEA, 63.9 moles, 3 equiv) were transferred into the reactor. The reactor was chilled to 10-15° C. and kept under a nitrogen atmosphere, after which 12.46 Kg of levulinic anhydride (58.11 moles, 3 eq) was charged into the reactor. When the addition was complete, the reaction was warmed to ambient temperature and stirred overnight or 12-16 hours. The completeness of the reaction was monitored by TLC (40:60 ethyl acetate/hexane) and by HPLC. 20 L of 10% citric acid, 10 L of water and 25 L of ethyl acetate were transferred into the reactor. This was stirred for 30 min and the layers were separated. The organic layer (EtOAc layer) was extracted with 20 L of water, 20 L 5% sodium bicarbonate and 20 L 25% brine solutions. The ethyl acetate solution was dried in 6-8 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The ED3 yield was 95%.

Synthesis of the EDC Trimer

Step 8. Preparation of ED4, Desilylation of ED3

A 100 L reactor was charged with 19720 g of 1-Silyl-2-Lev ED Dimer (ED3) (17.6 mol, 1 equiv) in 40 L of THF. The reactor was chilled to 0° C., after which 6903 g of tetrabutylammonium fluoride trihydrate (TBAF, 26.4 mol, 1.5 equiv) and 1511 mL (26.4 mol, 1.5 equiv) of glacial acetic acid were transferred into the reactor. When the addition was complete, the reaction was stirred and allowed to warm to ambient temperature. The reaction was monitored by TLC (40:60 ethyl acetate/hexane) and HPLC. The reaction required 3 hours to reach completion. When the reaction was completed, 20 L of water and 10 L of dichloromethane were transferred into the reactor and stirred for 30 min and the layers were separated. The organic layer (DCM layer) was extracted with 20 L water and 20 L 25% brine solutions. The dichloromethane solution was dried in 6-8 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The crude product was purified using a 200 L silica column using 140-200 L each of the following gradient profiles: 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 (EtOAc/heptane) and 200 L 100% EtOAc. The pure fractions were pooled and evaporated to a syrup and used in the next step. The isolated yield of ED4 was about 92%.

Synthesis of the EDC Trimer

Step 9. Preparation of ED5, TCA Formation

A 100 L reactor was charged with 14420 g of 1-OH-2-Lev ED Dimer (ED4) (16.35 mol, 1 equiv) in 30 L of dichloromethane. The reactor was purged with nitrogen and stirring was begun, after which 1222 mL of diazabicycloundecene (DBU, 8.175 mol, 0.5 equiv) and 23.61 Kg of trichloroacetonitrile (TCA, 163.5 mol, 10 equiv) were transferred into the reactor. Stirring was continued and the reaction was kept under nitrogen. After reagent addition, the reaction was allowed to warm to ambient temperature. The reaction was monitored by HPLC and TLC (40:60 ethyl acetate/heptane). The reaction took approximately 2 hours for reaction completion. When the reaction was completed, 20 L of water and 10 L of DCM were transferred into the reactor and stirred for 30 min and the layers were separated. The organic layer (DCM layer) was extracted with 20 L water and 20 L 25% brine solutions. The dichloromethane solution was dried in 6-8 Kg of anhydrous sodium sulfate. The solution was evaporated to a syrup (bath temp. 40° C.). The crude product was purified using a 200 L silica column using 140-200 L each of the following gradient profiles: 10:90, 20:80, 30:70, 40:60 and 50:50 (EtOAc/heptane). The pure fractions were pooled and evaporated to a syrup and used in the next step. The isolated yield of intermediate ED5 was about 70%.

Synthesis of the EDC Trimer

Step 10.

Preparation of EDC Trimer, Coupling of ED5 with Monomer C 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-benzyl (3-O-benzyl-2-O-levulinoyl)-β-D-glucopyranosyluronate-(1→4)-(3-O-acetyl-1,6-anhydro-2-azido)-2-deoxy-β-D-glucopyranose

A 100 L reactor was charged with 12780 g of 2-Lev 1-TCA ED Dimer (ED5) (7.38 mole, 1 eq., FW) and 4764 g of Monomer C (7.38 mole, 1 eq). The combined monomers were dissolved in 20 L toluene and co-evaporated at 40° C. Repeated was co-evaporation with an additional 2×20 L of toluene, after which 60 L of dichloromethane (DCM) was transferred into the reactor and dissolved. The reactor was purged with nitrogen and chilled to −20° C. When the temperature was between −20 and −10° C., 2962 g (11.2 moles, 0.9 eq) of TES Triflate were transferred into the reactor. After complete addition of TES Triflate the reaction was allowed to warm to 5° C. and stirring was continued. Completeness of the reaction was monitored by HPLC and TLC (35:65 ethyl acetate/Heptane). The reaction required 2-3 hours to reach completion. When the reaction was completed, 1133 g of triethylamine (TEA, 0.9 eq.) were transferred into the reactor and stirred for an additional 30 minutes. The organic layer (DCM layer) was extracted with 2×20 L 25% 4:1 sodium chloride/sodium bicarbonate solution. Dichloromethane solution was dried in 6-8 Kg of anhydrous sodium sulfate. The syrup was purified in a 200 L silica column using 140-200 L each of the following gradient profiles: 15:85, 20:80, 25:75, 30:70 and 35:65 (EtOAc/heptane). Pure fractions were pooled and evaporated to a syrup. The isolated yield of EDC Trimer was 48%. Formula: C₅₅H₆₀N₆O₁₈; Mol. Wt. 1093.09. The ¹H NMR spectrum (d6-acetone) of the EDC trimer is shown in FIG. 3.

Preparation of EDC1

Step 1:

Anhydro Ring Opening & Acetylation 6-O-acetyl-2-azido-2-deoxy-3,4-di-O-benzyl-α-D-glucopyranosyl-(1→4)-O-[benzyl 3-O-benzyl-2-O-levulinoyl-β-D-glucopyranosyluronate]-(1→4)-O-2-azido-2-deoxy-1,3,6-tri-O-acetyl-β-D-glucopyranose

7.0 Kg (6.44 mol) of EDC Trimer was dissolved in 18 L anhydrous Dichloromethane. 6.57 Kg (64.4 mol, 10 eq) of Acetic anhydride was added. The solution was cooled to −45 to −35° C. and 1.82 Kg (12.9 mol, 2 eq) of Boron Trifluoride etherate was added slowly. Upon completion of addition, the mixture was warmed to 0-10° C. and kept at this temperature for 3 hours until reaction was complete by TLC and HPLC. The reaction was cooled to −20° C. and cautiously quenched and extracted with saturated solution of sodium bicarbonate (3×20 L) while maintaining the mixture temperature below 5° C. The organic layer was extracted with brine (1×20 L), dried over anhydrous sodium sulfate, and concentrated under vacuum to a syrup. The resulting syrup of EDC1 (6.74 Kg) was used for step 2 without further purification. The ¹H NMR spectrum (d6-acetone) of the EDC-1 trimer is shown in FIG. 4.

Preparation of EDC2

Step 2:

Deacetylation 6-O-acetyl-2-azido-2-deoxy-3,4-di-O-benzyl-α-D-glucopyranosyl-(1→4)-O-[benzyl 3-O-benzyl-2-O-levulinoyl-β-D-glucopyranosyluronate]-(1→4)-O-2-azido-2-deoxy-3,6-di-O-acetyl-β-D-glucopyranose

The crude EDC1 product obtained from step 1 was dissolved in 27 L of Tetrahydrofuran and chilled to 15-20° C., after which 6 Kg (55.8 mol) of benzylamine was added slowly while maintaining the reaction temperature below 25° C. The reaction mixture was stirred for 5-6 hours at 10-15° C. Upon completion, the mixture was diluted with ethyl acetate and extracted and quenched with 10% citric acid solution (2×20 L) while maintaining the temperature below 25° C. The organic layer was extracted with 10% NaCl/1% sodium bicarbonate (1×20 L). The extraction was repeated with water (1×10 L), dried over anhydrous sodium sulfate and evaporated under vacuum to a syrup. Column chromatographic separation using silica gel yielded 4.21 Kg (57% yield over 2 steps) of EDC2[ also referred to as 1-Hydroxy-6-Acetyl EDC Trimer]. The ¹H NMR spectrum (d6-acetone) of the EDC-2 trimer is shown in FIG. 5.

Preparation of EDC3

Step 3:

Formation of TCA Derivative 6-O-acetyl-2-azido-2-deoxy-3,4-di-O-benzyl-α-D-glucopyranosyl-(1→4)-O-[benzyl 3-O-benzyl-2-O-levulinoyl-β-D-glucopyranosyluronate]-(1→4)-O-2-azido-2-deoxy-3,6-di-O-acetyl-1-O-trichloroacetimidoyl-β-D-glucopyranose

4.54 Kg (3.94 mol) of EDC2 was dissolved in 20 L of Dichloromethane. 11.4 Kg (78.8 mol, 20 eq) of Trichloroacetonitrile was added. The solution was cooled to −15 to −20° C. and 300 g (1.97 mol, 0.5 eq) of Diazabicycloundecene was added. The reaction was allowed to warm to 0-10° C. and stirred for 2 hours or until reaction was complete. Upon completion, water (20 L) was added and the reaction was extracted with an additional 10 L of DCM. The organic layer was extracted with brine (1×20 L), dried over anhydrous sodium sulfate, and concentrated under vacuum to a syrup. Column chromatographic separation using silica gel and 20-60% ethyl acetate/heptane gradient yielded 3.67 Kg (72% yield) of 1-TCA derivative, EDC3. The ¹H NMR spectrum (d6-acetone) of the EDC-3 trimer is shown in FIG. 6.

Preparation of EDCBA1

Step 4:

Coupling of EDC3 with BA Dimer Methyl O-6-O-acetyl-2-azido-2-deoxy-3,4-di-O-benzyl-α-D-glucopyranosyl)-(1→4)-O-[benzyl 3-O-benzyl-2-O-levulinoyl-β-D-glucopyranosyluronate]-(1→4)-O-2-azido-2-deoxy-3,6-di-O-acetyl-α-D-glucopyranosyl-(1→4)-O-[methyl 2-O-benzoyl-3-O-benzyl-α-L-Idopyranosyluronate]-(1→4)-2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-α-D-glucopyranoside

3.67 Kg (2.83 mol) of EDC3 and 3.16 Kg (3.96 mol, 1.4 eq) of BA Dimer was dissolved in 7-10 L of Toluene and evaporated to dryness. The resulting syrup was coevaporated with Toluene (2×15 L) to remove water. The dried syrup was dissolved in 20 L of anhydrous Dichloromethane, transferred to the reaction flask, and cooled to −15 to −20° C. 898 g (3.4 mol, 1.2 eq) of triethylsilyl triflate was added while maintaining the temperature below −5° C. When the addition was complete, the reaction was immediately warmed to −5 to 0° C. and stirred for 3 hours. The reaction was quenched by slowly adding 344 g (3.4 mol, 1.2 eq) of Triethylamine. Water (15 L) was added and the reaction was extracted with an additional 10 L of DCM. The organic layer was extracted with a 25% 4:1 Sodium Chloride/Sodium Bicarbonate solution (2×20 L), dried over anhydrous sodium sulfate, and evaporated under vacuum to a syrup. The resulting syrup of the pentasaccharide, EDCBA1 was used for step 5 without further purification. The ¹H NMR spectrum (d6-acetone) of the EDCBA-1 pentamer is shown in FIG. 7.

Preparation of EDCBA2

Step 5:

Hydrolysis of Levulinyl moiety Methyl O-6-O-acetyl-2-azido-2-deoxy-3,4-di-O-benzyl-α-D-glucopyranosyl)-(1→4)—O-[benzyl 3-O-benzyl-β-D-glucopyranosyluronate]-(1→4)-O-2-azido-2-deoxy-3,6-di-O-acetyl-α-D-glucopyranosyl)-(1→4)-O-[methyl 2-O-benzoyl-3-O-benzyl-α-L-Idopyranosyluronate]-(1→4)-2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-α-D-glucopyranoside

The crude EDCBA1 from step 4 was dissolved in 15 L of Tetrahydrofuran and chilled to −20 to −25° C. A solution containing 679 g (13.6 mol) of Hydrazine monohydrate and 171 g (1.94 mol) of Hydrazine Acetate in 7 L of Methanol was added slowly while maintaining the temperature below −20° C. When the addition was complete, the reaction mixture was allowed to warm to 0-10° C. and stirred for several hours until the reaction is complete, after which 20 L of Ethyl acetate was added and the reaction was extracted with 10% citric acid (2×12 L). The organic layer was washed with water (1×12 L), dried over anhydrous sodium sulfate, and evaporated under vacuum to a syrup. Column chromatographic separation using silica gel and 10-45% ethyl acetate/heptane gradient yielded 2.47 Kg (47.5% yield over 2 steps) of EDCBA2. The ¹H NMR spectrum (d6-acetone) of the EDCBA-2 pentamer is shown in FIG. 8.

Preparation of EDCBA Pentamer

Step 6:

THP Formation Methyl O-6-O-acetyl-2-azido-2-deoxy-3,4-di-O-benzyl-α-D-glucopyranosyl-(1→4)-O-[benzyl 3-O-benzyl-2-O-tetrahydropyranyl-β-D-glucopyranosyluronate]-(1→4)-O-2-azido-2-deoxy-3,6-di-O-acetyl-α-D-glucopyranosyl-(1→4)-O-[methyl 2-O-benzoyl-3-O-benzyl-α-L-Idopyranosyluronate]-(1→4)-2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-α-D-glucopyranoside

2.47 Kg (1.35 mol) of EDCBA2 was dissolved in 23 L Dichloroethane and chilled to 10-15° C., after which 1.13 Kg (13.5 mol, 10 eq) of Dihydropyran and 31.3 g (0.135 mol, 0.1 eq) of Camphorsulfonic acid were added. The reaction was allowed warm to 20-25° C. and stirred for 4-6 hours until reaction was complete. Water (15 L) was added and the reaction was extracted with an additional 10 L of DCE. The organic layer was extracted with a 25% 4:1 Sodium Chloride/Sodium Bicarbonate solution (2×20 L), dried over anhydrous sodium sulfate, and evaporated under vacuum to a syrup. Column chromatographic separation using silica gel and 10-35% ethyl acetate/heptane gradient yielded 2.28 Kg (88.5% yield) of fully protected EDCBA Pentamer. The ¹H NMR spectrum (d6-acetone) of the EDCBA pentamer is shown in FIG. 9.

Preparation of API1

Step 1:

Saponification Methyl O-2-azido-2-deoxy-3,4-di-O-benzyl-α-D-glucopyranosyl-(1→4)-O-3-O-benzyl-2-O-tetrahydropyranyl-β-D-glucopyranosyluronosyl-(1→4)-O-2-azido-2-deoxy-α-D-glucopyranosyl-(1→4)-O-3-O-benzyl-α-L-Idopyranosyluronosyl-(1→4)-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranoside disodium salt

To a solution of 2.28 Kg (1.19 mol) of EDCBA Pentamer in 27 L of Dioxane and 41 L of Tetrahydrofuran was added 45.5 L of 0.7 M (31.88 mol, 27 eq) Lithium hydroxide solution followed by 5.33 L of 30% Hydrogen peroxide. The reaction mixture was stirred for 10-20 hours to remove the acetyl groups. Then, 10 L of 4 N (40 mol, 34 eq) sodium hydroxide solution was added. The reaction was allowed to stir for an additional 24-48 hours to hydrolyze the benzyl and methyl esters completely. The reaction was then extracted with ethyl acetate. The organic layer was extracted with brine solution and dried with anhydrous sodium sulfate. Evaporation of the solvent under vacuum gave a syrup of API1 [also referred to as EDCBA(OH)₅] which was used for the next step without further purification.

Preparation of API2

Step 2:

O-Sulfonation Methyl O-2-azido-2-deoxy-3,4-di-O-benzyl-6-O-sulfo-α-D-glucopyranosyl-(1→4)-O-3-O-benzyl-2-O-tetrahydropyranyl-β-D-glucopyranosyluronosyl-(1→4)-O-2-azido-2-deoxy-3,6-di-O-sulfo-α-D-glucopyranosyl-(1→4)-O-3-O-benzyl-2-O-sulfo-α-L-idopyranuronosyl-(1→4)-2-azido-2-deoxy-6-O-sulfo-α-D-glucopyranoside, heptasodium salt

The crude product of API1 [aka EDCBA(OH)₅] obtained in step 1 was dissolved in 10 L Dimethylformamide. To this was added a previously prepared solution containing 10.5 Kg (66 moles) of sulfur trioxide-pyridine complex in 10 L of Pyridine and 25 L of Dimethylformamide. The reaction mixture was heated to 50° C. over a period of 45 min. After stiffing at 1.5 hours at 50° C., the reaction was cooled to 20° C. and was quenched into 60 L of 8% sodium bicarbonate solution that was kept at 10° C. The pH of the quench mixture was maintained at pH 7-9 by addition of sodium bicarbonate solution. When all the reaction mixture has been transferred, the quench mixture was stirred for an additional 2 hours and pH was maintained at pH 7 or greater. When the pH of quench has stabilized, it was diluted with water and the resulting mixture was purified using a preparative HPLC column packed with Amberchrom CG161-M and eluted with 90%-10% Sodium Bicarbonate (5%) solution/Methanol over 180 min. The pure fractions were concentrated under vacuum and was then desalted using a size exclusion resin or gel filtration (Biorad) G25 to give 1581 g (65.5% yield over 2 steps) of API2 [also referred to as EDCBA(OSO₃)₅]. The ¹H NMR spectrum (d6-acetone) of API-2 pentamer is shown in FIG. 10.

Preparation of API3

Step 3:

Hydrogenation Methyl O-2-amino-2-deoxy-6-O-sulfo-α-D-glucopyranosyl-(1→4)-O-2-O-tetrahydropyranyl-β-D-glucopyranosyluronosyl-(1→4)-O-2-amino-2-deoxy-3,6-di-O-sulfo-α-D-glucopyranosyl-(1→4)-O-2-O-sulfo-α-L-idopyranuronosyl-(1→4)-2-amino-2-deoxy-6-O-sulfo-α-D-glucopyranoside, heptasodium salt

A solution of 1581 g (0.78 mol) of O-Sulfated pentasaccharide API2 in 38 L of Methanol and 32 L of water was treated with 30 wt % of Palladium in Activated carbon under 100 psi of Hydrogen pressure at 60-65° C. for 60 hours or until completion of reaction. The mixture was then filtered through 1.0μ and 0.2μ filter cartridges and the solvent evaporated under vacuum to give 942 g (80% yield) of API3 [also referred to as EDCBA(OSO₃)₅(NH₂)₃]. The ¹H NMR spectrum (d6-acetone) of API-3 pentamer is shown in FIG. 11.

Preparation of Fondaparinux Sodium

Step 4:

N-Sulfation & Removal of THP Methyl O-2-deoxy-6-O-sulfo-2-(sulfoamino)-α-D-glucopyranosyl-(1→4)—O-β-D-glucopyranuronosyl-(1→4)-O-2-deoxy-3,6-di-O-sulfo-2-(sulfoamino)-α-D-glucopyranosyl-(1→4)-O-2-O-sulfo-α-L-idopyranuronosyl-(1→4)-2-deoxy-6-O-sulfo-2-(sulfoamino)-α-D-glucopyranoside, decasodium salt

To a solution of 942 g (0.63 mol) of API3 in 46 L of water was slowly added 3.25 Kg (20.4 mol, 32 eq) of Sulfur trioxide-pyridine complex, maintaining the pH of the reaction mixture at pH 9-9.5 during the addition using 2 N sodium hydroxide solution. The reaction was allowed to stir for 4-6 hours at pH 9.0-9.5. When reaction was complete, the pH was adjusted to pH 7.0 using 50 mM solution of Ammonium acetate at pH 3.5. The resulting N-sulfated EDCBA(OSO₃)₅(NHSO₃)₃ mixture was purified using Ion-Exchange Chromatographic Column (Varian Preparative 15 cm HiQ Column) followed by desalting using a size exclusion resin or gel filtration (Biorad G25). The resulting mixture was then treated with activated charcoal and the purification by ion-exchange and desalting were repeated to give 516 g (47.6% yield) of the purified Fondaparinux Sodium form.

Analysis of the Fondaparinux sodium identified the presence of P1, P2, P3, and P4 in the fondaparinux. P1, P2, P3, and P4 were identified by standard analytical methods.

INTERMEDIATES

The monomers used in the processes described herein may be prepared as described in the art, or can be prepared using the methods described herein.

The synthesis of Monomer A-2 (CAS Registry Number 134221-42-4) has been described in the following references: Arndt et al., Organic Letters, 5(22), 4179-4182, 2003; Sakairi et al., Bulletin of the Chemical Society of Japan, 67(6), 1756-8, 1994; and Sakairi et al., Journal of the Chemical Society, Chemical Communications, (5), 289-90, 1991, and the references cited therein, which are hereby incorporated by reference in their entireties.

Monomer C(CAS Registry Number 87326-68-9) can be synthesized using the methods described in the following references: Ganguli et al., Tetrahedron: Asymmetry, 16(2), 411-424, 2005; Izumi et al., Journal of Organic Chemistry, 62(4), 992-998, 1997; Van Boeckel et al., Recueil: Journal of the Royal Netherlands Chemical Society, 102(9), 415-16, 1983; Wessel et al.,Helvetica Chimica Acta, 72(6), 1268-77, 1989; Petitou et al., U.S. Pat. No. 4,818,816 and references cited therein, which are hereby incorporated by reference in their entireties.

Monomer E (CAS Registry Number 55682-48-9) can be synthesized using the methods described in the following literature references: Hawley et al., European Journal of Organic Chemistry, (12), 1925-1936, 2002; Dondoni et al., Journal of Organic Chemistry, 67(13), 4475-4486, 2002; Van der Klein et al., Tetrahedron, 48(22), 4649-58, 1992; Hori et al., Journal of Organic Chemistry, 54(6), 1346-53, 1989; Sakairi et al., Bulletin of the Chemical Society of Japan, 67(6), 1756-8, 1994; Tailler et al.,Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio–Organic Chemistry, (23), 3163-4, (1972-1999) (1992); Paulsen et al., Chemische Berichte, 111(6), 2334-47, 1978; Dasgupta et al., Synthesis, (8), 626-8, 1988; Paulsen et al., Angewandte Chemie, 87(15), 547-8, 1975; and references cited therein, which are hereby incorporated by reference in their entireties.

Monomer B-1 (CAS Registry Number 444118-44-9) can be synthesized using the methods described in the following literature references: Lohman et al., Journal of Organic Chemistry, 68(19), 7559-7561, 2003; Orgueira et al., Chemistry—A European Journal, 9(1), 140-169, 2003; Manabe et al., Journal of the American Chemical Society, 128(33), 10666-10667, 2006; Orgueira et al., Angewandte Chemie, International Edition, 41(12), 2128-2131, 2002; and references cited therein, which are hereby incorporated by reference in their entireties.
Synthesis of Monomer D
Monomer D was prepared in 8 synthetic steps from glucose pentaacetate using the following procedure:

Pentaacetate SM-B was brominated at the anomeric carbon using HBr in acetic acid to give bromide derivative IntD1. This step was carried out using the reactants SM-B, 33% hydrogen bromide, acetic acid and dichloromethane, stirring in an ice water bath for about 3 hours and evaporating at room temperature. IntD1 was reductively cyclized with sodium borohydride and tetrabutylammonium iodide in acetonitrile using 3 Å molecular sieves as dehydrating agent and stirring at 40° C. for 16 hours to give the acetal derivative, IntD2. The three acetyl groups in IntD2 were hydrolyzed by heating with sodium methoxide in methanol at 50° C. for 3 hours and the reaction mixture was neutralized using Dowex 50WX8-100 resin (Aldrich) in the acid form to give the trihydroxy acetal derivative IntD3.

The C4 and C6 hydroxyls of IntD3 were protected by mixing with benzaldehyde dimethyl acetate and camphor sulphonic acid at 50° C. for 2 hours to give the benzylidene-acetal derivative IntD4. The free hydroxyl at the C3 position of IntD4 was deprotonated with sodium hydride in THF as solvent at 0° C. and alkylated with benzyl bromide in THF, and allowing the reaction mixture to warm to room temperature with stirring to give the benzyl ether IntD5. The benzylidene moiety of IntD5 was deprotected by adding trifluoroacetic acid in dichloromethane at 0° C. and allowing it to warm to room temperature for 16 hours to give IntD6 with a primary hydroxyl group. IntD6 was then oxidized with TEMPO (2,2,6,6-tetramethyl-1-piperidine-N-oxide) in the presence of tetrabutylammonium chloride, sodium bromide, ethyl acetate, sodium chlorate and sodium bicarbonate, with stirring at room temperature for 16 hours to form the carboxylic acid derivative IntD7. The acid IntD7 was esterified with benzyl alcohol and dicyclohexylcarbodiimide (other reactants being hydroxybenzotriazole and triethylamine) with stirring at room temperature for 16 hours to give Monomer D.

Synthesis of the BA Dimer

The BA Dimer was prepared in 12 synthetic steps from Monomer B1 and Monomer A2 using the following procedure:

The C4-hydroxyl of Monomer B-1 was levulinated using levulinic anhydride and diisopropylethylamine (DIPEA) with mixing at room temperature for 16 hours to give the levulinate ester BMod1, which was followed by hydrolysis of the acetonide with 90% trifluoroacetic acid and mixing at room temperature for 4 hours to give the diol BMod2. The C1 hydroxyl of the diol BMod2 was silylated with tert-butyldiphenylsilylchloride by mixing at room temperature for 3 hours to give silyl derivative BMod3. The C2-hydroxyl was then benzoylated with benzoyl chloride in pyridine, and mixed at room temperature for 3 hours to give compound BMod4. The silyl group on BMod4 was then deprotected with tert-butyl ammonium fluoride and mixing at room temperature for 3 hours to give the C1-hydroyl BMod5. The C1-hydroxyl is then allowed to react with trichloroacetonitrile in the presence of diazobicycloundecane (DBU) and mixing at room temperature for 2 hours to give the trichloroacetamidate (TCA) derivative BMod6, which suitable for coupling, for example with Monomer A-2.

Monomer A-2 was prepared for coupling by opening the anhydro moiety with BF₃.Et₂O followed by acetylation of the resulting hydroxyl groups to give the triacetate derivative AMod1.

Monomer A2 was prepared for the coupling reaction by opening the anhydro moiety and acetylation of the resulting hydroxyl groups to give the triacetate derivative AMod1. This transformation occurs using boron trifluoride etherate, acetic anhydride and dichloromethane, between −20° C. and room temperature for 3 hours. The C1-Acetate of AMod1 was then hydrolyzed and methylated in two steps to give the diacetate AMod3. That is, first AMod1 was reacted with trimethylsilyl iodide and mixed at room temperature for 2 hours, then reacted with and tetrabutyl ammonium iodide. This mixture was reacted with diisoproylethylamine and methanol and stirred for 16 hours at room temperature, thus forming AMod3. The C4 and C6 acetates of AMod3 are hydrolyzed with sodium methoxide to give the diol Amod4. The AMod3 mixture was also subjected to mixing at room temperature for 3 hours with Dowex 50 Wx4x8-100 resin in the acid form for neutralization. This formed Amod4. The C6-hydroxyl of AMod4 is then benzoylated by treating with benzoyl chloride in pyridine at −40° C. and then allowing it to warm up to −10° C. over 2 hours to give AMod5.

Coupling of monomer AMod5 with the free C4-hydroxyl group of BMod6 was performed in the presence of BF₃.Et₂O and dichloromethane with mixing between −20° C. and room temperature for 3 hours to provide disaccharide BA1. The C4-levulinyl moiety of the disaccharide was then hydrolyzed with hydrazine to give the BA Dimer, which is suitable for subsequent coupling reactions.

Synthesis of EDC Trimer

The EDC Trimer was prepared in 10 synthetic steps from Monomer E, Monomer D and Monomer C using the following procedure:

Monomer E was prepared for coupling by opening the anhydro moiety with BF₃.Et₂O followed by acetylation of the resulting hydroxyl groups to give diacetate EMod1. This occurs by the addition of Monomer E with boron trifluoride etherate, acetic anhydride and dichloromethane at −10° C., and allowing the reaction to warm to room temperature with stirring for 3 hours. The C1-Acetate of EMod1 is then hydrolyzed to give the alcohol, EMod2. This occurs by reacting Emod1 with hydrazine acetate and dimethylformamide and mixing at room temperature for 3 hours. The C1-hydroxyl of Emod2 is then reacted with trichloroacetonitrile to give the trichloro acetamidate (TCA) derivative EMod3 suitable for coupling, which reaction also employs diazabicycloundecane and dichloromethane and mixing at room temperature for 2 hours.

Monomer D, having a free C4-hydroxyl group, was coupled with monomer EMod3 in the presence of triethylsilyl triflate with mixing at −40° C. for 2 hours to give the disaccharide ED Dimer. The acetal on ring sugar D of the ED Dimer is hydrolyzed to give the C1,C2-diol ED1. This occurs by reacting the ED Dimer with 90% trifluoro acetic acid and mixing at room temperature for 4 hours. The C1-hydroxyl moiety of ED1 was then silylated with tert-butyldiphenylsilyl chloride to give the silyl derivative ED2. The C2-hydroxyl of ED2 was then allowed to react with levulinic anhydride in the presence of dimethylaminopyridine (DMAP) and diethylisopropylamine for approximately 16 hours to give the levulinate ester ED3. The TBDPS moiety is then deprotected by removal with tert-butylammonium fluoride in acetic acid with mixing at room temperature for 3 hours to give ED4 having a C1-hydroxyl. The C1-hydroxyl moiety of ED4 was then allowed to react with trichloroacetonitrile to give the TCA derivative ED5, which is suitable for coupling.

The C1-hydroxyl moiety of ED4 is then allowed to react with trichloroacetonitrile to give the TCA derivative ED5 suitable for coupling using diazabicycloundecane and dichloromethane, and mixing at room temperature for 2 hours. Monomer C, havinga free C4-hydroxyl group, was then coupled with the disaccharide ED5 in the presence of triethylsilyl triflate and mixed at −20° C. for 2 hours to give the trisaccharide EDC Trimer.

Synthesis of the EDCBA Pentamer

The EDCBA Pentamer was prepared using the following procedure:

The preparation of EDCBA Pentamer is accomplished in two parts as follows. In part 1, the EDC Trimer, a diacetate intermediate, is prepared for the coupling reaction with Dimer BA by initially opening the anhydro moiety and acetylation of the resulting hydroxyl groups to give the tetraacetate derivative EDC1. This occurs by reacting the EDC Trimer with boron trifluoride etherate, acetic anhydride and dichlormethane and stirring between −10° C. and room temperature for 3 hours. The C1-Acetate of EDC1 is then hydrolyzed to give the alcohol, EDC2, by reacting EDC1 with benzylamine [BnNH₂] and tetrahydrofuran and mixing at −10° C. for 3 hours. The C1-hydroxyl of EDC2 is then reacted with trichloroacetonitrile and diazabicycloundecane, with mixing at room temperature for 2 hours, to give the trichloro acetamidate (TCA) derivative EDC3 suitable for coupling.

In Part 2 of the EDCBA Pentameter synthesis, the Dimer BA, having a free C4-hydroxyl group, is coupled with trisaccharide EDC3 in the presence of triethylsilyltriflate at −30° C. mixing for 2 hours to give the pentasaccharide EDCBA1. The levulinyl ester on C2 of sugar D in EDCBA1 is hydrolyzed with a mixture of deprotecting agents, hydrazine hydrate and hydrazine acetate and stiffing at room temperature for 3 hours to give the C2-hydroxyl containing intermediate EDCBA2. The C2-hydroxyl moiety on sugar D of EDCBA2 is then alkylated with dihydropyran (DHP) in the presence of camphor sulfonic acid (CSA) and tetrahydrofuran with mixing at room temperature for 3 hours to give the tetrahydropyranyl ether (THP) derivative, EDCBA Pentamer.

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A fast and effective hydrogenation process of protected pentasaccharide: A key step in the synthesis of fondaparinux sodium, Org Process Res Dev 2013, 17: 869, http://pubs.acs.org/doi/full/10.1021/op300367c

An improved method for the simultaneous removal of O-benzyl and N-carboxybenzyl groups as well as reducing azide groups to amines in protected heparin-like pentasaccharides, a key process in fondaparinux sodium synthesis, is reported. Under catalytic transfer hydrogenation conditions, using readily available and inexpensive ammonium formate, the hydrogenolysis is done in less than an hour in good yield and purity. This procedure represents a major advantage over the previously published procedures, the latter of which involve several hours/days of hydrogenation reaction under catalytic reduction using gaseous hydrogen.

Synthesis of Compound 1 (FONDAPARINUX)

………………

SYNTHESIS

WO2013003001A1

US20130005954

In the synthesis of Fondaparinux sodium, the monomers XII, XVIII, XXVII, XXXVIII, XXXXI and dimers XIX, XX, XL described herein may be made either by processes described in the art or, by a process as described herein. The XII and XVIII monomers may then linked to form a disaccharide XX, XXXIX and XXVII monomers may then linked to form a disaccharide XL, XLIII and XX dimers may then linked to form a tetrasaccharide, XLVII tetramer and XLV monomer may be linked to form a pentasaccharide (XLVIII) pentamer. The XLVIII pentamer is an intermediate that may be converted through a series of reactions to fondaparinux sodium. This strategy described herein provides an efficient method for multi-kilogram preparation of fondaparinux in high yields and highly stereoselective purity.

Fondaparinux sodium (LIII) was prepared in 3 synthetic steps from O – S pentasaccharide (L) using the following procedure:

Fondaparinux Sodium (LIII)

Preparation of Fondaparinux sodium (LIII)—

N- sulfonation of Deprotected Pentasaccharide (LI) methyl 0-2-deoxy-3,6-di-0- sulfo-2-(sulfoamino)-a-D-glucopyranosyl-(l— >4)-0-2-0-sulfo-a-L- idopyranurosyl-( 1— >4)-2-deoxy-6-0-sulfo-2-(sulfoamino)-a-D-glucopyranoside,decasodium salt

A solution of deprotected pentasaccharide (LI) (145 gm) in water (2.54 V) was adjusted to a pH of 9.5 – 10.5 with 1 N NaOH solution. S03-pyridine complex (156 gm) was added into 3 lots every 15 min, the pH being maintained at 9.5-10.5 by automatic addition of 1 N NaOH. The mixture was stirred for 2 hrs at RT, during this aqueous NaOH (IN solution) was added to maintain pH at 9.5 – 10.5. After neutralization to pH 7 – 7.5 by addition of HC1 solution, the mixture was evaporated using vacuum. The residue was dissolved in water (1.6 L) at RT, to this solution was added acetone (1.6 L) at RT. The reaction mass was cooled to 5°C – 1 0 °C and stirred for 1 hr. The solid was filtered and washed with cold acetone: water (1 :1). The clear filtrate was distilled off completely under vacuum below 55°C. The residue was dissolved in water (1.6 L) at RT, and to this solution was added acetone(1.6 L) at RT. The mixture was cooled to 5 to 10°C and stirred for 1 hr. The solid was filtered and washed with cold acetone/water (1 :1). The clear filtrate was distilled off completely under vacuum below 55°C. The residue was dissolved in water (0.7 L) and charcoal (40 gm) was added at RT. The mixture was stirred for 30 min at RT then filtered. To the filtrate was added charcoal (40 gm) at RT. The mixture was stirred for 30 min at RT then filtered. To the filtrate was added charcoal (40 gm) at RT. The mixture was stirred for 30 min at RT then filtered. The pH of the clear filtrate was adjusted to 8.0 – 8.5 with IN NaOH solution and distilled off completely under vacuum below 55 °C. The residue was dissolved in 0.5 M NaCl solution and layered onto a column of Dowex^® 1×2 -400 resins using a gradient of NaCl solution (0.5 to 10M). The product fractions were combined and distilled off under vacuum below 55 °C up to 1 – 2 L volume. The solid was filtered off and the clear filtrate was distilled off under vacuum below 55 °C up to slurry stage and subjected to azeotropic distillation with methanol two times. The solid residue was stirred with methanol (2.13 L) at RT for 1 hr and the solid was filtered off and washed with methanol. The wet solid was again stirred with methanol (2.13 L) at RT for 1 hr and the solid was filtered off and washed with methanol. The wet solid was again stirred with methanol (2.13 L) at RT for 1 hr and the solid was filtered off and washed with methanol. The above solid was dissolved in water and the pH adjusted to 4 – 4.5 with IN HC1 solution and charcoalized three times with 26 gm of charcoal at RT for 15-30 minutes and filtered off. To the clear filtrate was added 0.39 kg of NaCl, then methanol was added (35 volume) at RT and the mixture was stirred for 15-30 minutes. The solution was decanted and the sticky mass was stirred with methanol (0.65 L) at RT for 15-30 minutes. The solid was filtered off and dissolved in water, and the pH adjusted to 8 – 8.5 with IN NaOH solution. The solution was filtered through 0.45 micron paper & distilled off completely under vacuum below 55°C. The solution was subjected to azeotropic distillation with methanol to give highly pure fondaparinux sodium (97.17 gm) (HPLC purity 99.7%).

SOR Results

Three batches of product made in accordance with the present processes provided the following stereoisomeric optical rotation results:

Specification: Between +50.0° and +60.0°.

Batch- 1 = +55.1°

Batch-2 = +55.7° Batch-3 = +55.4°.

INTERMEDIATES

Synthetic Procedures

The following abbreviations are used herein: Ac is acetyl; MS is molecular sieve; DMF is dimethyl formamide; Bn is benzyl; MDC is dichloromethane; THF is tetrahydrofuran; TFA is trifluoro acetic acid; MeOH is methanol; RT is room temperature; Ac₂O is acetic anhydride; HBr is hydrogen bromide; EtOAc is ethyl acetate; Cbz is benzyloxycarbonyl; CADS is chloro acetyl disaccharide; HDS is hydroxy disaccharide; NMP is N-methylpyrrolidone.

Methyl 3-O-benzyl-4-O-monochloro acetyl-β-L-idopyranuronate

Route of Synthesis for α-Methyl-6-o-acetyl-3-o-benzyl-2-(benzyloxy carbonyl)amino-2-deoxy-α-D-glucopyranoside

Methyl 6-O-acetyl-3-O-benzyl-2-(benzyloxy carbonyl)amino-2-deoxy-4-O-(methyl-2-O acetyl-3-O-benzyl-α-L-idopyranosyluronate)-glucopyranoside

Route of Synthesis for 1,6-Anhydro-2-azido-3-O-acetyl-2-deoxy-beta-D-glucopyranose

Route of synthesis for Methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranuronate

Route of synthesis for 3-O-Acetyl-1,6-anhydro-2-azido-4-O-2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyl methyluronate-beta-D-glucopyranose

(or)

3-O-Acetyl-1,6-anhydro-2-azido-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-beta-D-glucopyranose

Route of Synthesis for 1,6-Anhydro-2-azido-3,4-di-O-benzyl-2-deoxy-beta-D-glucopyranose

Synthesis of Disaccharide XLIII

Disaccharide XLIII was prepared in 2 synthetic steps from CADS sugar (XL) using the following procedure:

CADS sugar XL was acetylated at the anomeric carbon using AC₂O and TFA to give acetyl derivative XLII. This step was carried out using the reactants CADS, AC₂O and TFA, stirring in an ice water bath for about 5-24 hours, preferably 20 hours, and evaporating to residue under vacuum. Residue was recrystallized in ether. Acetyl CADS (XLII) was brominated at the anomeric carbon using titanium tetra bromide in MDC andethylacetate and stirring at 20° C.-50° C. for 6-16 hours, preferably 6 hours, to give the bromo derivative, (XLIII) after work-up and recrystallization from solvent/alcohol.

Synthesis of the Monosaccharide (XLV)

The monosaccharide (XLV) was prepared in 2 synthetic steps from monomer (XLI) using the following procedure:

Mono sugar (XLI) was acetylated at the anomeric carbon using AC₂O and TFA to give acetyl derivative (XLIV). This step was carried out using the reactants Mono sugar (XLI), AC₂O and TFA, stirring in an ice water bath for about 5-24 hours, preferably 24 hours, and evaporating to residue under vacuum. Residue was recrystallized in ether. Acetyl Mono sugar (XLIV) was brominated at the anomeric carbon using titanium tetra bromide in MDC and ethyl acetate and stirring at 20° C.-50° C. for 6-20 hours, preferably 16 hours, to give the bromo derivative, (XLV) after work-up and recrystallization from ether.

Synthesis of the Hydroxy Tetrasaccharide (XLVII)

The hydroxy tetrasaccharide (XLVII) was prepared in 2 synthetic steps from disaccharide (XLIII) and HDS (XX) using the following procedure:

Disaccharide (XLIII), was coupled with disaccharide (XX) in the presence of silver carbonate, silver per chlorate and 4 A° MS in MDC and stirred at ambient temperature for 5-12 hrs, preferably 4-6 hours, in the dark followed by work-up and purification in water/methanol to give the tetrasaccharide (XLVI). The d echloroacetylation of tetrasaccharide (XLVI) was carried out in THF, ethanol and pyridine in the presence of thiourea at reflux for 6 to 20 hrs, preferably 12 hours, to give the hydroxy tetrasaccharide (XLVIII).

Synthesis of the Pentasaccharide (XLVIII)

The pentasaccharide (XLVIII) was prepared in 2 synthetic steps from monosaccharide (XLV) and tetrasaccharide (XLVII) using the following procedure:

Monosaccharide (XLV), was coupled with tetrasaccharide (XLVII) in the presence of 2,4,6-collidine, silver triflate and 4 A° MS in MDC and stirred at −10° C. to −20° C. for 1 hr in the dark followed by work-up and purification by column chromatography to give the pentasaccharide (XLVIII).

Synthesis of OS Pentasaccharide (L)

The OS pentasaccharide (L) was prepared in 2 synthetic steps from pentasaccharide (XLVIII) using the following procedure:

Pentasaccharide (XLVIII) was deacetylated in the presence of NaOH in mixture of solvents of MDC, methanol and water at 0° C. to 35° C., for 1-2 hrs followed by work-up and distillation to obtain deacetylated pentasaccharide (XLIX) which was subjected to O-sulfonation in DMF in the presence of SO₃-trimethylamine (TMA) at 50° C. to 100° C., preferably 50° C.-55° C., for 6-24 hrs, preferably 12 hours, followed by salt removal through Sephadex® resin and column chromatography purification, then pH adjustment by dilute NaOH to give OS pentasaccharide (L).

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INTERMEDIATE

WO2013011460A1

highly pure 4-Ο-β-ϋ- glucopyranosyl- 1 ,6-anhydro- -D-glucopyranose

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SYNTHESIS

WO2013115817A1

Synthesis of Fondaparinux

Fondaparinux was prepared using the following procedure:

Conversion of FPP (also referred to a Fully Protected Pentamer) to FondaparinuxSodium:

Reagents: 1. NaOH, H₂0₂, LiOH, Dioxane, RT, 24-48 h; 2. Py.S0₃, DMF, 60°C, 2h, CG-161 purification; 3. 10% Pd/C, H₂, 72h; 4. (a) Py.S0₃, NaOH, NH₄OAc, 12h, (b) HiQ NH₄OAc/ NaCl ion-exchange, Sephadex Desalt and (c) HiQ NaCl ion-exchange, Sephadex Desalt. The ester moieties in EDCBA Pentamer-CB were hydrolyzed with sodium and lithium hydroxide in the presence of hydrogen peroxide in dioxane mixing at room temperature for 24- 48 hours to give the pentasaccharide intermediate API1-CB. The five hydroxyl moieties in API1-CB were sulfated using a pyridine-sulfur trioxide complex in dimethylformamide, mixing at 60°C for 2 hours and then purified using column chromatography (CG-161), to give the pentasulfated pentasaccharide API2-CB. The intermediate API2-CB was then hydrogenated to reduce the three azides on sugars E, C and A to amines and the reductive deprotection of the six benzyl ethers to their corresponding hydroxyl groups to form the intermediate API3-CB. This transformation occurs by reacting API2-CB with 10% palladium/carbon catalyst with hydrogen gas for 72 hours. The three amines on API3-CB were then sulfated using the pyridine-sulfur trioxide complex in sodium hydroxide and ammonium acetate, allowing the reaction to proceed for 12 hours . The crude fondaparinux is purified and is subsequently converted to its salt form. The crude mixture was purified using an ion-exchange chromatographic column (HiQ resin) followed by desalting using a size exclusion resin or gel filtration (Biorad Sephadex G25) to give the final product, fondaparinux sodium.

Preparation of Fondaparinux Sodium – Step 4: N-Sulfation of API-3-CB:

Methyl 0-2-deoxy-6-0-sulfo-2-(sulfoamino)-a-D-glucopyranosyl-(l→4)-0^-D- glucopyranuronosyl-(l→4)-0-2-deoxy-3,6-di-0-sulfo-2-(sulfoamino)-a-D-glucopyranosyl- (l→4)-0-2-0-sulfo-a-L-idopyranuronosyl-(l→4)-2-deoxy-6-0-sulfo-2-(sulfoamino)-a-D- glucopyranoside, decasodium salt

To a solution of 25.4 gram (16.80 mmol, leq) of API-3-CB in 847 mL of water was slowly added 66.85 gram (446.88 mmol, 25eq) of sulfur trioxide-pyridine complex, maintaining the pH of the reaction mixture at pH 9-9.5 during the addition using 2N sodium hydroxide solution. The reaction was allowed to stir for 4 hours at pH 9.0 – 9.5. When reaction was completed, the pH was adjusted 7.0 by using 70 mL of 50 mmol Ammonium acetate solution pH -3.5. The resulting N-Sulfated Cellobiose mixture was purified using Ion-Exchange

Chromatographic Column followed by desalting using size exclusion resin to gave gram ( %) of the purified Fondaparinux Sodium form.

To a solution of 942 g (0.63 mol) of API3 in 46 L of water was slowly added 3.25 Kg (20.4 mol, 32 eq) of Sulfur trioxide-pyridine complex, maintaining the pH of the reaction mixture at pH 9-9.5 during the addition using 2 N sodium hydroxide solution. The reaction was allowed to stir for 4-6 hours at pH 9.0-9.5. When reaction was complete, the pH was adjusted to pH 7.0 using 50 mM solution of Ammonium acetate at pH 3.5. The resulting N- sulfated EDCBA(OS0₃)5(NHS0₃)3 mixture was purified using Ion-Exchange Chromatographic Column (Varian Preparative 15 cm HiQ Column) followed by desalting using a size exclusion resin or gel filtration (Biorad G25). The resulting mixture was then treated with activated charcoal and the purification by ion-exchange and desalting were repeated to give 516 g (47.6% yield) of the purified Fondaparinux sodium form.

INT

SCHEME 1 – Synthesis of Monomer A-2 & AMod5 fBuildinq Block Al

Reagents: 1. NaOMe, MeOH, RT, 2hr, 50wx resin; 2. (Bu₃Sn)₂0 (0.8equiv), ACN, MS, reflux, 3h; 3.l₂ (1.5 equiv), 5°C to RT, 2h; 4. NaH (2 equiv), DMF, p-MeOC₆H₄CH₂Br (PMB-Br, 2.5 equiv), -20°C to RT, 2h; 5. NaN₃, DMF, 120°C, 12h; 6. NaH, DMF, BnBr, 0°C to RT, 3h.; 7. BF₃.Et₂0, Ac₂0, DCM, -20°C to RT, 3h; 8. (a) TMS-I, TBAI, RT, 2h; (b) DIPEA, MeOH, 16h, RT; 9. NaOMe, Dowex 50WX8-100 resin H+ form, RT, 3h; 10. Pyridine, Bz-CI, -40°C to -10°C, 2h;

Scheme 2 – Synthesis of Monomer B-1 and BMod6 fBuildinq Block B1

Reagents: 1. NaH, BnBr, THF, DMF, 0° to 65°C, 3h; 2. 66% Acetic Acid/H₂0, 40 °C, 16h; 3. Nal0₄, (Bu)₄NBr, DCM, H₂0, Dark, 3h; 4. (PhS)₃CH, n-BuLi, THF, -78 °C, 3h; 5. CuCI₂/CuO, MeOH, H₂0, 3h; 6. 90% TFA/H₂0, DCM, RT, 2h; 7. DMF, CSA 2-methoxypropene, 0° to RT, 16hrs; MeOH, TEA. 8. Lev₂0, DIPEA, RT, 16h; 9. 90% TFA, RT, 4h; 10. Imidazole, TBDPSi-CI, RT, 3h; 11. Pyridine, BzCI, RT, 3h; 12. TBAF, RT, 3h; 13. TCA, DBU, RT, 2h; Also see, e.g., Bonnaffe et al., Tetrahedron Lett., 41, 307-311, 2000; Bonnaffe et al., Carbohydr. Res., 2003, 338, 681-686, 2003; and Seeberger et al., J. Org. Chem., 2003, 68, 7559- 7561, 2003.

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Carbohydrate Research, 2012 ,  vol. 361, p. 155 – 161

¹H NMR (D₂O) δ: 5.68 (d, J = 3.8 Hz, 1H, H-1D), 5.56 (d, J = 3.4 Hz, 1H, H-1F), 5.24 (d, J = 3.8 Hz, 1H, H-1G), 5.07 (d, J = 3.5 Hz, 1H, H-1H), 4.68 (d, J = 7.9 Hz, 1H, H-5G), 4.54 (dd, J = 11.4, 2.2 Hz, 1H, H-1E), 4.48-4.34 (m, 6H, H-6F, 6H, 6’H, 6D, 3E, 2G), 4.33-4.30 (m, 1H, H-6’F), 4.25-4.17 (m, 4H, H-4G, 3G, 6’D, 5F), 4.06-3.98 (m, 2H, H-4F, 5H), 3.94 (dd, J = 9.7, 2.2 Hz, 1H, H-5D), 3.92-3.86 (m, 2H,H-3E, H-4E), 3.85-3.80 (m, 2H, H-5E, 4H), 3.73-3.60 (m, 3H, H-3H, 3D, 4D), 3.53-3.44 (m, 2H, H-2F, H-2E), 3.47 (s, 3H, OMe), 3.34 (dd, J = 10.2, 3.7 Hz, 1H, H-2H), 3.31(dd, J = 10.2, 3.7 Hz, 1H, H-2D)

FONDAPARINUX

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Synthesis of intermediates

US8288515

Synthetic Procedures

The following abbreviations are used herein: Ac is acetyl; ACN is acetonitrile; MS is molecular sieves; DMF is dimethyl formamide; PMB is p-methoxybenzyl; Bn is benzyl; DCM is dichloromethane; THF is tetrahydrofuran; TFA is trifluoro acetic acid; CSA is camphor sulfonic acid; TEA is triethylamine; MeOH is methanol; DMAP is dimethylaminopyridine; RT is room temperature; CAN is ceric ammonium nitrate; Ac₂O is acetic anhydride; HBr is hydrogen bromide; TEMPO is tetramethylpiperidine-N-oxide; TBACl is tetrabutyl ammonium chloride; EtOAc is ethyl acetate; HOBT is hydroxybenzotriazole; DCC is dicyclohexylcarbodiimide; Lev is levunlinyl; TBDPS is tertiary-butyl diphenylsilyl; TCA is trichloroacetonitrile; O-TCA is O-trichloroacetimidate; Lev₂O is levulinic anhydride; DIPEA is diisopropylethylamine; Bz is benzoyl; TBAF is tetrabutylammonium fluoride; DBU is diazabicycloundecane; BF₃.Et₂O is boron trifluoride etherate; TMSI is trimethylsilyl iodide; TBAI is tetrabutylammonium iodide; TES-Tf is triethylsilyl trifluoromethanesulfonate (triethylsilyl triflate); DHP is dihydropyran; PTS is p-toluenesulfonic acid.

The monomers used in the processes described herein may be prepared as described in the art, or can be prepared using the methods described herein.

The synthesis of Monomer A-2 (CAS Registry Number 134221-42-4) has been described in the following references: Arndt et al., Organic Letters, 5(22), 4179-4182, 2003; Sakairi et al., Bulletin of the Chemical Society of Japan, 67(6), 1756-8, 1994; and Sakairi et al., Journal of the Chemical Society, Chemical Communications, (5), 289-90, 1991, and the references cited therein, which are hereby incorporated by reference in their entireties.

Monomer C(CAS Registry Number 87326-68-9) can be synthesized using the methods described in the following references: Ganguli et al., Tetrahedron: Asymmetry, 16(2), 411-424, 2005; Izumi et al., Journal of Organic Chemistry, 62(4), 992-998, 1997; Van Boeckel et al., Recueil: Journal of the Royal Netherlands Chemical Society, 102(9), 415-16, 1983; Wessel et al.,Helvetica Chimica Acta, 72(6), 1268-77, 1989; Petitou et al., U.S. Pat. No. 4,818,816 and references cited therein, which are hereby incorporated by reference in their entireties.

Monomer E (CAS Registry Number 55682-48-9) can be synthesized using the methods described in the following literature references: Hawley et al., European Journal of Organic Chemistry, (12), 1925-1936, 2002; Dondoni et al., Journal of Organic Chemistry, 67(13), 4475-4486, 2002; Van der Klein et al., Tetrahedron, 48(22), 4649-58, 1992; Hori et al., Journal of Organic Chemistry, 54(6), 1346-53, 1989; Sakairi et al., Bulletin of the Chemical Society of Japan, 67(6), 1756-8, 1994; Tailler et al.,Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio–Organic Chemistry, (23), 3163-4, (1972-1999) (1992); Paulsen et al., Chemische Berichte, 111(6), 2334-47, 1978; Dasgupta et al., Synthesis, (8), 626-8, 1988; Paulsen et al., Angewandte Chemie, 87(15), 547-8, 1975; and references cited therein, which are hereby incorporated by reference in their entireties.

Monomer B-1 (CAS Registry Number 444118-44-9) can be synthesized using the methods described in the following literature references: Lohman et al., Journal of Organic Chemistry, 68(19), 7559-7561, 2003; Orgueira et al., Chemistry—A European Journal, 9(1), 140-169, 2003; Manabe et al., Journal of the American Chemical Society, 128(33), 10666-10667, 2006; Orgueira et al., Angewandte Chemie, International Edition, 41(12), 2128-2131, 2002; and references cited therein, which are hereby incorporated by reference in their entireties.
Synthesis of Monomer D
Monomer D was prepared in 8 synthetic steps from glucose pentaacetate using the following procedure:

Pentaacetate SM-B was brominated at the anomeric carbon using HBr in acetic acid to give bromide derivative IntD1. This step was carried out using the reactants SM-B, 33% hydrogen bromide, acetic acid and dichloromethane, stirring in an ice water bath for about 3 hours and evaporating at room temperature. IntD1 was reductively cyclized with sodium borohydride and tetrabutylammonium iodide in acetonitrile using 3 Å molecular sieves as dehydrating agent and stirring at 40° C. for 16 hours to give the acetal derivative, IntD2. The three acetyl groups in IntD2 were hydrolyzed by heating with sodium methoxide in methanol at 50° C. for 3 hours and the reaction mixture was neutralized using Dowex 50WX8-100 resin (Aldrich) in the acid form to give the trihydroxy acetal derivative IntD3.

The C4 and C6 hydroxyls of IntD3 were protected by mixing with benzaldehyde dimethyl acetate and camphor sulphonic acid at 50° C. for 2 hours to give the benzylidene-acetal derivative IntD4. The free hydroxyl at the C3 position of IntD4 was deprotonated with sodium hydride in THF as solvent at 0° C. and alkylated with benzyl bromide in THF, and allowing the reaction mixture to warm to room temperature with stirring to give the benzyl ether IntD5. The benzylidene moiety of IntD5 was deprotected by adding trifluoroacetic acid in dichloromethane at 0° C. and allowing it to warm to room temperature for 16 hours to give IntD6 with a primary hydroxyl group. IntD6 was then oxidized with TEMPO (2,2,6,6-tetramethyl-1-piperidine-N-oxide) in the presence of tetrabutylammonium chloride, sodium bromide, ethyl acetate, sodium chlorate and sodium bicarbonate, with stirring at room temperature for 16 hours to form the carboxylic acid derivative IntD7. The acid IntD7 was esterified with benzyl alcohol and dicyclohexylcarbodiimide (other reactants being hydroxybenzotriazole and triethylamine) with stirring at room temperature for 16 hours to give Monomer D.

Synthesis of the BA Dimer

The BA Dimer was prepared in 12 synthetic steps from Monomer B1 and Monomer A2 using the following procedure:

The C4-hydroxyl of Monomer B-1 was levulinated using levulinic anhydride and diisopropylethylamine (DIPEA) with mixing at room temperature for 16 hours to give the levulinate ester BMod1, which was followed by hydrolysis of the acetonide with 90% trifluoroacetic acid and mixing at room temperature for 4 hours to give the diol BMod2. The C1 hydroxyl of the diol BMod2 was silylated with tert-butyldiphenylsilylchloride by mixing at room temperature for 3 hours to give silyl derivative BMod3. The C2-hydroxyl was then benzoylated with benzoyl chloride in pyridine, and mixed at room temperature for 3 hours to give compound BMod4. The silyl group on BMod4 was then deprotected with tert-butyl ammonium fluoride and mixing at room temperature for 3 hours to give the C1-hydroyl BMod5. The C1-hydroxyl is then allowed to react with trichloroacetonitrile in the presence of diazobicycloundecane (DBU) and mixing at room temperature for 2 hours to give the trichloroacetamidate (TCA) derivative BMod6, which suitable for coupling, for example with Monomer A-2.

Monomer A-2 was prepared for coupling by opening the anhydro moiety with BF₃.Et₂O followed by acetylation of the resulting hydroxyl groups to give the triacetate derivative AMod1.

Monomer A2 was prepared for the coupling reaction by opening the anhydro moiety and acetylation of the resulting hydroxyl groups to give the triacetate derivative AMod1. This transformation occurs using boron trifluoride etherate, acetic anhydride and dichloromethane, between −20° C. and room temperature for 3 hours. The C1-Acetate of AMod1 was then hydrolyzed and methylated in two steps to give the diacetate AMod3. That is, first AMod1 was reacted with trimethylsilyl iodide and mixed at room temperature for 2 hours, then reacted with and tetrabutyl ammonium iodide. This mixture was reacted with diisoproylethylamine and methanol and stirred for 16 hours at room temperature, thus forming AMod3. The C4 and C6 acetates of AMod3 are hydrolyzed with sodium methoxide to give the diol Amod4. The AMod3 mixture was also subjected to mixing at room temperature for 3 hours with Dowex 50 Wx4x8-100 resin in the acid form for neutralization. This formed Amod4. The C6-hydroxyl of AMod4 is then benzoylated by treating with benzoyl chloride in pyridine at −40° C. and then allowing it to warm up to −10° C. over 2 hours to give AMod5.

Coupling of monomer AMod5 with the free C4-hydroxyl group of BMod6 was performed in the presence of BF₃.Et₂O and dichloromethane with mixing between −20° C. and room temperature for 3 hours to provide disaccharide BA1. The C4-levulinyl moiety of the disaccharide was then hydrolyzed with hydrazine to give the BA Dimer, which is suitable for subsequent coupling reactions.

Synthesis of EDC Trimer

The EDC Trimer was prepared in 10 synthetic steps from Monomer E, Monomer D and Monomer C using the following procedure:

Monomer E was prepared for coupling by opening the anhydro moiety with BF₃.Et₂O followed by acetylation of the resulting hydroxyl groups to give diacetate EMod1. This occurs by the addition of Monomer E with boron trifluoride etherate, acetic anhydride and dichloromethane at −10° C., and allowing the reaction to warm to room temperature with stirring for 3 hours. The C1-Acetate of EMod1 is then hydrolyzed to give the alcohol, EMod2. This occurs by reacting Emod1 with hydrazine acetate and dimethylformamide and mixing at room temperature for 3 hours. The C1-hydroxyl of Emod2 is then reacted with trichloroacetonitrile to give the trichloro acetamidate (TCA) derivative EMod3 suitable for coupling, which reaction also employs diazabicycloundecane and dichloromethane and mixing at room temperature for 2 hours.

Monomer D, having a free C4-hydroxyl group, was coupled with monomer EMod3 in the presence of triethylsilyl triflate with mixing at −40° C. for 2 hours to give the disaccharide ED Dimer. The acetal on ring sugar D of the ED Dimer is hydrolyzed to give the C1,C2-diol ED1. This occurs by reacting the ED Dimer with 90% trifluoro acetic acid and mixing at room temperature for 4 hours. The C1-hydroxyl moiety of ED1 was then silylated with tert-butyldiphenylsilyl chloride to give the silyl derivative ED2. The C2-hydroxyl of ED2 was then allowed to react with levulinic anhydride in the presence of dimethylaminopyridine (DMAP) and diethylisopropylamine for approximately 16 hours to give the levulinate ester ED3. The TBDPS moiety is then deprotected by removal with tert-butylammonium fluoride in acetic acid with mixing at room temperature for 3 hours to give ED4 having a C1-hydroxyl. The C1-hydroxyl moiety of ED4 was then allowed to react with trichloroacetonitrile to give the TCA derivative ED5, which is suitable for coupling.

The C1-hydroxyl moiety of ED4 is then allowed to react with trichloroacetonitrile to give the TCA derivative ED5 suitable for coupling using diazabicycloundecane and dichloromethane, and mixing at room temperature for 2 hours. Monomer C, having a free C4-hydroxyl group, was then coupled with the disaccharide ED5 in the presence of triethylsilyl triflate and mixed at −20° C. for 2 hours to give the trisaccharide EDC Trimer.

Synthesis of the EDCBA Pentamer

The EDCBA Pentamer was prepared using the following procedure:

The preparation of EDCBA Pentamer is accomplished in two parts as follows. In part 1, the EDC Trimer, a diacetate intermediate, is prepared for the coupling reaction with Dimer BA by initially opening the anhydro moiety and acetylation of the resulting hydroxyl groups to give the tetraacetate derivative EDC1. This occurs by reacting the EDC Trimer with boron trifluoride etherate, acetic anhydride and dichlormethane and stirring between −10° C. and room temperature for 3 hours. The C1-Acetate of EDC1 is then hydrolyzed to give the alcohol, EDC2, by reacting EDC1 with benzylamine [BnNH₂] and tetrahydrofuran and mixing at −10° C. for 3 hours. The C1-hydroxyl of EDC2 is then reacted with trichloroacetonitrile and diazabicycloundecane, with mixing at room temperature for 2 hours, to give the trichloro acetamidate (TCA) derivative EDC3 suitable for coupling.

In Part 2 of the EDCBA Pentameter synthesis, the Dimer BA, having a free C4-hydroxyl group, is coupled with trisaccharide EDC3 in the presence of triethylsilyltriflate at −30° C. mixing for 2 hours to give the pentasaccharide EDCBA1. The levulinyl ester on C2 of sugar D in EDCBA1 is hydrolyzed with a mixture of deprotecting agents, hydrazine hydrate and hydrazine acetate and stiffing at room temperature for 3 hours to give the C2-hydroxyl containing intermediate EDCBA2. The C2-hydroxyl moiety on sugar D of EDCBA2 is then alkylated with dihydropyran (DHP) in the presence of camphor sulfonic acid (CSA) and tetrahydrofuran with mixing at room temperature for 3 hours to give the tetrahydropyranyl ether (THP) derivative, EDCBA Pentamer.

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Intermediates listed  on the internet

Fondaparinux sodium Intermediates

Fondaparinux sodium N-4

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Fondaparinux sodium N-3

114903-05-8

a-D-Glucopyranoside, Methyl O-2-azido-2-deoxy-3,4-bis-O-(phenylMethyl)-a-D-glucopyranosyl-(14) -O-2,3-bis-O-(phenylMethyl)-b-D-glucopyranuronosyl-(14)-O-2-azido- 2-deoxy-a-D-glucopyranosyl-(14)-O-3-O-(phenylMethyl)-a-L-idopyranu ronosyl-(14)-2-deoxy-2

FSC

114903-05-8

Chemical Name: O-[methyl2,3-di-O-benzyl-4-O-chloroacetyl-beta-Dglucopyranosyluronate]-( 1-4)-3-O-acetyl-1,6-anhydro-2-azido-2-deoxy-beta-D-glucopyranose

Description

CAS number 87907-02-6 Synonym O-[methyl2,3-di-O-benzyl-4-O-chloroacetyl-beta-Dglucopyranosyluronate]-(1-4)-3-O-acetyl-1,6-anhydro-2-azido-2-deoxy-beta-D-glucopyranose Molecular Formula C31H34ClN3O12 Molecular Weight 676.07

Chemical Name: 1,6-anhydro-2-azido-3,4-di-O-benzyl-2-deoxy-ß-D-glucopyranose

Description

CAS number 443916-61-8 Synonym 1,6-anhydro-2-azido-3,4-di-O-benzyl-2-deoxy-ß-D-glucopyranose Molecular Formula C20H21N3O4 Molecular Weight 367.4

Chemical Name: Methyl-6-O-acetyl-3-O-benzyl-2(benzyloxycarbonyl) amino-2-deoxy-4-O-(methyl2-O-acetyl-3-O-benzyl-alfa-L-idopyranosyl uronate)-alfa-D-glucopyranoside

Description

CAS number 114869-97-5 Synonym Methyl-6-O-acetyl-3-O-benzyl-2(benzyloxycarbonyl) amino-2-deoxy-4-O-(methyl2-O-acetyl-3-O-benzyl-alfa-L-idopyranosyl uronate)-alfa-D-glucopyranoside Molecular Formula C40H47NO15 Molecular Weight 781.8

Chemical Name: Benzyl-6-O-acetyl-3-O-benzyl-2-(benzyloxycarbonyl)amino-2-deoxy-4-O-(methyl2-Oacetyl-3-O-benzyl-alfa-L-idopyranosyluronate)-alfa-D-glucopyranoside

Description

CAS number 87907-11-7 Synonym Benzyl-6-O-acetyl-3-O-benzyl-2-(benzyloxycarbonyl)amino-2-deoxy-4-O-(methyl2-Oacetyl-3-O-benzyl-alfa-L-idopyranosyluronate)-alfa-D-glucopyranoside Molecular Formula C46H51NO15 Molecular Weight 857.33

Chemical Name: 3-O-Benzyl-1,2-O-isopropylidene-alpha-D-Glucofurasone

Description

CAS number 22529-61-9 Synonym 3-O-Benzyl-1,2-O-isopropylidene-alpha-D-Glucofurasone Molecular Formula C16H22O6

Chemical Name: Tetrasaccharide, ( Please refer Synonym )

Description

CAS number N-A Synonym Methyl-O-6-methyl-2,3-di-O-benzyl-beta-D-glucopyranouronosyl-(1->4)-3’6di-O’acetyl-2-azido-2-dexoy-alfa-D-glucopyranosyl-(1->4)-2-O-acetyl-3-O-benzyl-6-methyl-alfa-L-idopyranourinosyl-(1->4)-6-O-acetyl3-O-be nzyI-2-(benzyIoxycarbo n yl)amino-2-deoxy-alfa-D-gIucopyranoside Molecular Formula C71H82N4027 Molecular Weight 1423.42

Chemical Name: Fondaparinux Sodium N-3 Intermediate

Description

CAS number 114903-05-8 Synonym MethylO-(2-azido-3,4-di-O-benzyl-2-deoxy-a-D-glucopyranosyl)-(1-4)-O-(2,3-di-Obenzyl-ß-D-glucopyranosyluronicacid)-(1-4)-O-(2-azido-2-deoxy-a-D-glucopyranosyl)-(1-4)-O-(3-O-benzyl-a-L-idopyranosyluronic acid)-(1-4)-3-O-benzyl-2-benzyloxycarbonylamino-2-deoxy-a-D-glucopyranoside,N-3 Intermediate, Fondaparinux Molecular Formula C81H91N7O27 Molecular Weight 1593.60

References

FONDAPARINUX

• Arixtra home page
• Alchemia home page

The three specialties available in the United States – dalteparin (Fragmin, Pfizer), enoxaparin (Lovenox, Sanofi-Aventis) and tinzaparin (Innohep, Bristol-Myers Squibb) – the first two are found in Brazil, enoxaparin under the names Lovenox, Cutenox and Dripanina.