Topiroxostat

Xanthine oxidase inhibitor

FOR GOUT AND HYPERURICEMIA

Launched – 2013, Fuji YakuhinSanwa, Topiloric  Uriadec

IUPAC Name: 4-(5-pyridin-4-yl-1H-1,2,4-triazol-3-yl)pyridine-2-carbonitrile

CAS Registry Number: 577778-58-6

4 – [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazol-3 - yl] pyridine-2 – carbonitrile (1)

5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole

3-(3-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole
Synonyms: 4-(5-PYRIDIN-4-YL-1H-1,2,4-TRIAZOL-3-YL)PYRIDINE-2-CARBONITRILE,

AC1NRB9T, Topiroxostat (JAN/INN),  DB01685, D09786, FYX-051
SK-0910

4-[5-PYRIDIN-4-YL-1H-[1,2,4]TRIAZOL-3-YL]-PYRIDINE-2-CARBONITRILE,

C₁₃H₈N_{6 MF,}248.2482 MW

TOPIROXOSTAT

FYX-051, TOPIROXOSTAT is a xanthine oxidase inhibitor. This agent was approved in Japan by Fuji Yakuhin and Sanwa for the treatment of gout and hyperuricemia in 2013 and launched at the same year. In 2009, the compound was licensed to Sanwa by Fuji Yakuhin in Japan for the codevelopment and commercialization of gout.

The number of patients with hyperuricemia in Japan is reported to be 1.25 million and the number suffering from asymptomatic hyperuricemia is estimated to reach several millions. Hyperuricemia is becoming a popular disease.

Presently, hyperuricemia and gout due to hyperuricemia are treated by improving the living environment and administering various drug therapies for each period when an attack of gout is predicted to occur (presymptomatic period), when an attack of gout occurs, or when an attack of gout subsides. That is, preventive therapy is conducted in the presymptomatic period by administering colchicines as well as controlling the daily living environment. When an attack occurs, drug therapy using non-steroidal or steroidal anti-inflammatory agents is mainly conducted. After the attack subsides, patients are given guidance to improve their lifestyle. When improvement is judged insufficient, an assessment is made as to whether hyperuricemia is caused by reduced excretion of uric acid or by increased production of uric acid followed by treatment with drugs, which exhibit a uricosuric effect, such as probenecid and benzbromarone, those which inhibit resorption of uric acid, such as sulfinpyrazone, those which improve acidurea conditions, such as citrates, and xanthine oxidase inhibitors which inhibit production of uric acid, such as allopurinol. Colchicine is said to be able to prevent about 90% of attacks through inhibiting chemotaxis and phagocytosis of leukocytes, such as neutrophils, if administration thereof has been completed within a few hours before the attack. Since colchicine has various adverse effects, however, the use thereof is limited to the minimum and it is therefore difficult to timely administer it.

Accordingly, drug therapies are mainly adopted, but only allopurinol is available for the treatment of a disease caused by increased production of uric acid. However, a metabolite of allopurinol, oxypurinol, tends to accumulate and may cause calculi formation. Furthermore, this drug has been reported to induce adverse events such as rash, a decreased renal function and hepatitis, and it is not easy to administer.

Examples of compounds having xanthine oxidase inhibiting activity that can be used for treating gout caused by increased production of uric acid and that are effective for hyperuricemia and gout due to hyperuricemia have been described in J. Medicinal Chemistry, 1975, Vol. 18, No. 9, pp. 895–900, Japanese Patent Publication No. 49-46622 and Japanese Patent Publication No. 50-24315, which disclose some 1,3,5-substituted or 3,5-substituted 1,2,4-triazole compounds.

4 – [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazol-3 - yl] pyridine-2 – carbonitrile (1) has a xanthine oxidase inhibitory activity and serum uric acid level known as the agent that reduces (Patent Document 1).

The method for producing the compound (1), for example, 2 by Reissert Henze reaction isonicotinic acid methyl N-oxide – is a cyano isonicotinate, and the hydrazide which is then, 4 – this condensed cyanopyridine After obtaining a hydrazide of isonicotinic acid N-oxide (Patent Document 1, Example 12) and method, a cyano group after introduction, 4 by Reissert Henze reaction – method of condensing a cyano pyridine is known (Patent Document 1, Example 39).Further, 4 – as a starting material cyano-N-oxide, a triazole ring after construction (Patent Document 3), Reissert Henze unprotected or (Patent Document 2) to protect the ring condensed with isonicotinic acid hydrazide method of obtaining the compound (1) by introducing a cyano group by the reaction have also been reported.

The crystalline polymorph, yet the same molecule with the same chemical composition, the molecular arrangement in the crystal are different, and are different crystalline states. The pharmaceutical compounds having crystal polymorphism such the differences in physicochemical properties, affect pharmacological activity, solubility, bioavailability, stability and the like are known.Therefore, when the crystal polymorphism is present in a pharmaceutically useful compound, producing compounds of the crystalline form highly useful from polymorphs thereof is desirable.

However, 4 of the above Patent Document – no description about the presence of crystalline polymorph on carbonitrile – pyridine-2-[yl 5 - (pyridin-4 - yl)-1H-1, 2,4 - - -3 triazol] It has not been, to these manufacturing methods, it is disclosed a method for the purpose of improving the chemical purity and yield, there is no description of the crystallographic plane.

Method of producing topiroxostat, useful for preventing or treating gout; and its intermediates. Picks up from WO2012060308, claiming the use of this topiroxostat for treating renal dysfunction. Along with the concurrently published WO2014017515, claiming crystalline Forms I and II of this compound, which, Fuji Yakuhin, in collaboration with Sanwa Kagaku, has developed and launched for the treatment of gout and hyperuricemia.WO-2014017516

Crystalline Forms I and II of topiroxostat, useful for preventing or treating gout. Along with the concurrently published WO2014017516, claiming a method of producing this compound. Picks up from WO2012060308, claiming a method of treating renal dysfunction using topiroxostat, which Fuji Yakuhin, in collaboration with Sanwa Kagaku, has developed and launched for the treatment of gout and hyperuricemia.WO-2014017515

•  novel 1,2,4-triazole compounds having an optionally substituted 2-cyanopyridin-4-yl group at 3-position and an optionally substituted aromatic group at 5-position inhibit a xanthine oxidase and are useful for treatment of gout and hyperuricemia, and have previously filed a patent application (Patent Document 1). The compounds can be prepared according to a method shown by the following reaction scheme:
• wherein TMS represents trimethylsilyl group and Ar represents an aromatic group.
• Although this method can achieve the object in a small-scale production, there were such problems that the process for production of a substituted or unsubstituted 2-cyanoisonicotinic acid hydrazide is complicated, and a reaction solvent must be selected in compliance with the physical property of the product compound in each step, and isolation of a product is required in each step. Furthermore, the overall yield is not sufficiently high, and therefore there is a problem in the production on an industrial scale.
  Patent Document 1: JP-A-2002-017825
• • A compound represented by formula (1) which is a starting material may be prepared by a method described in, for example, JP-A-47-7120, JP-A-61-152661A, JP-A-62-149673, JP-A-2002-528447, or European Patent Application No. 559363 specification. However, it is preferable to prepare compound (1) according to the following reaction scheme:

…………………………………

EP1650204A1

Example 2

Preparation of 5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole p-toluenesulfonate
• To the toluene solution obtained in Example 1 (2) was added 2-propanol (700 mL), and the mixture was stirred. To the resulting solution was added p-toluenesulfonic acid monohydrate (151.16 g) and the resulting mixture was stirred for 8 hours at an internal temperature of 80°C. The mixture was brought to room temperature, and the precipitated crystals were taken out and washed with 2-propanol (210 mL×2). The white crystals were dried under reduced pressure at 60°C for 15 hours to give 106.0 g of the captioned compound as white crystals. Subsequently, 90.0 g of the crystals was suspended in a mixture of 2-butanol (49 mL) and water (491 mL) and heated to an internal temperature of 80°C for 1 hour. The internal temperature was brought to room temperature, and the crystals were filtered and washed with a mixture of 2-butanol and water (1:10) (270 mL×3). The resulting crystals were dried under reduced pressure at 60°C for 15 hours to give 75.7 g of the captioned compound in a high purity.
• ¹H―NMR(DMSO-d₆)δppm:2.29(s,3H), 7.11 (m,2H), 7.48 (dd, 2H, J=6.48, 1.62Hz) , 8.32-8.35(m, 3H) , 8.57(dd, 1H, J=1.62, 0.81Hz) , 8.94-8.98(m, 3H)

Example 3

Preparation of 5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole

• To the white crystals (50.5g) obtained in Example 2 was added 2-propanol (937.5 mL) and water (312.5 mL), and the resulting mixture was heated and dissolved at an internal temperature of 80°C. Immediately thereafter, the solution was filtered and the filtrate was cooled to an internal temperature of 20°C. To the resulting suspension was added dropwise 0.52 mol/l of an aqueous sodium hydrogen carbonate solution (250 mL), and the mixture was stirred at room temperature for 2 hours. Then the crystals were filtered and washed with water (150 mL×3) and 2-butanol (150 mL×2). The crystals were dried under reduced pressure at 80°C for 15 hours to give 29.4 g of the captioned compound as pale yellow crystals.
• ¹H―NMR(DMSO-d₆)δppm:8.02(dd, 2H, J=4.59, 1.62Hz),8.32(dd, 1H, J=5.13, 1.62Hz), 8.55(dd, 1H, J=1.62, 1.08Hz), 8.80(dd, 2H, J=4.59, 1.62Hz), 8.93 (dd, 1H, J=5.13, 1.08Hz)

………………………………

SYNTHESIS

US7074816

Example 12

5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole

1) Production of methyl isonicotinate N-oxide

13.9 g of isonicotinic acid N-oxide was added to 209 ml of methylene chloride, 29.7 g of 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline was further added thereto, and the mixture was stirred under argon atmosphere at room temperature for one hour. 32.1 g of methanol was added to this mixture, which was stirred at room temperature for 17 hours. After the solvent was evaporated under reduced pressure, the residue was subjected to silica gel column chromatography. Chloroform-acetone (3:1) was used as an eluent to yield 11.1 g of a white powder.

¹H-NMR (CDCl₃) δppm: 3.95 (3H, s), 7.88 (2H, d, J=7.25 Hz), 8.22 (2H, J=7.25 Hz)

2) Production of Methyl 2-cyanoisonicotinate

11.1 g of the crystal obtained in 1) was dissolved in 170 ml of acetonitrile, 14.6 g of triethylamine and 21.5 g of trimethylsilylnitrile were added thereto, and the mixture was refluxed under argon atmosphere for 16 hours. After the solvent was evaporated under reduced pressure, the residue was subjected to silica gel column chromatography. Chloroform-acetone (95:5) was used as an eluent to yield 8.44 g of a pale yellow powder.

¹H-NMR (CDCl₃) δppm: 4.01 (3H, s), 8.08 (1H, d, J=5.45 Hz), 8.24 (1H, s), 8.90 (1H, d, J=5.45 Hz)

3) Production of 2-cyanoisonicotinic acid hydrazide

8.44 g of the crystal obtained in 2) was added to 85 ml of methanol, 1.84 g of hydrazine was further added thereto, and the mixture was stirred under argon temperature for 2 hours. After the solvent was evaporated under reduced pressure, chloroform was added to the residue, which was stirred at room temperature for one hour. The precipitated crystal was filtered, washed with chloroform and dried with a vacuum pump to yield 4.15 g of a pale yellow powder.

¹H-NMR (DMSO-d₆) δppm: 4.72 (2H, s), 8.05 (1H, d, J=5.12 Hz), 8.31 (1H, s),8.90 (1H, d, J=5.12 Hz), 10.23 (1H, s)

4) Production of the Object Compound

2.67 g of 4-cyanopyridine was dissolved in 40 ml of methanol, 0.83 g of sodium methoxide was added thereto, and the mixture was stirred at room temperature for one hour. Then 4.15 g of the crystal obtained in 3) was added and the mixture was refluxed for 37 hours. After the reaction completed, the precipitated solid was filtered, washed with methanol and dried with a vacuum pump to yield 3.66 g of the object compound as a yellow powder.

¹H-NMR (DMSO-d₆) δppm: 8.01 (2H, dd, J=4.54, 1.57 Hz), 8.31 (1H, dd, J=5.11, 1.65 Hz), 8.53 (1H, dd, J=1.65, 0.50 Hz), 8.80 (2H, dd, J=4.54, 1.57 Hz), 8.93 (1H, dd, J=5.11, 0.50 Hz)

Example 39

5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole

1) Production of isonicotinic acid (N-2-tert-butoxycarbonyl)hydrazide-1-oxide

585 ml of methylene chloride was added to 39.0 g of isonicotinic acid N-oxide, and after 34.0 g of triethylamine was further added thereto, the mixture was cooled under argon atmosphere to −15° C. 33.5 g of ethyl chlorocarbonate in 117 ml of methylene chloride was added dropwise to this mixture, which was stirred at a temperature from −5 to −10° C. for one hour. Then 44.4 g of tert-butyl ester of carbamic acid in 117 ml of methylene chloride was added dropwise to this mixture and it was allowed to slowly rise to room temperature while it was stirred. The precipitated solid was filtered after 15 hours, washed with methylene chloride, and dried with a vacuum pump to yield 49.7 g of white crystal.

¹H-NMR (DMSO-d₆) δppm: 1.42 (9H, s), 7.82 (2H, d, J=7.09 Hz), 8.33 (2H, d, J=7.09 Hz), 9.02 (1H, s), 10.44 (1H, s)

Production of 2-cyanoisonicotinic acid hydrazine 1½ P-Toluenesulfonic acid salt

228 ml of dioxane was added to 30.4 g of the crystal obtained in 1), and after 13.1 g of trimethylsilyl cyanide and 38.8 g of N,N-dimethylcarbamoyl chloride were further added thereto, the mixture was stirred under argon atmosphere at 60° C. for 5 hours. After the solvent was evaporated under reduced pressure, the residue was dissolved in ethyl acetate and subsequently washed with 1.5 M sodium carbonate aqueous solution and a saturated saline solution and dried over magnesium sulfate. After the magnesium sulfate was filtered off, the solvent was evaporated under reduced pressure. Ethyl acetate was added to the residue, 68.5 g of p-toluenesulfonic acid monohydrate was added thereto, and the mixture was stirred at room temperature for 22 hours. The precipitated crystal was filtered, washed with ethyl acetate, and dried with a vacuum pump to yield 40.3 g of white crystal 2).

¹H-NMR (DMSO-d₆) δppm: 2.28 (4.5H, s), 7.12 (3H, dd, J=7.92 & 0.66 Hz), 7.48 (3H, dd, J=7.92 & 0.66 Hz), 8.10 (1H, dd, J=5.11 & 1.81 Hz), 8.39 (1H, dd, J=1.81 & 0.33 Hz), 8.99 (1H, dd, J=5.11 & 0.33 Hz)

3) Production of 5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole

9.98 g of 4-cyanopyridine was dissolved in 250 ml of methanol, and after 7.77 g of sodium methoxide was added thereto, the mixture was stirred at room temperature for one hour. Then 40.3 g of the crystal obtained in 2) was added and the mixture was refluxed for 24 hours. After the reaction completed, the precipitated crystal was filtered, washed with methanol, and dried with a vacuum pump to yield 16.3 g of yellow crystal.

¹H-NMR (DMSO-d₆) δppm: 8.01 (2H, dd, J=4.54 & 1.57 Hz), 8.31 (1H, dd, J=5.11 & 1.65 Hz), 8.53 (1H, dd, J=1.65 & 0.50 Hz), 8.80 (2H, dd, J=4.54 & 1.57 Hz), 8.93 (1H, dd, J=5.11 & 0.50 Hz)

4) Production of 5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole

45 ml of ethanol and 15 ml of 1-methyl-2-pyrrolidone were added to 3.0 g of the crystal obtained in 3), and the mixture was heated and stirred at 80° C. for 19 hours. The crystal was filtered, subsequently washed with a mixture of ethanol and 1-methyl-2-pyrrolidone (3:1) and ethanol, and dried with a vacuum pump to yield 2.71 g of yellow crystal.

5) Production of 5-(2-cyano-4-pyridyl)-3-(4-pyridyl)-1,2,4-triazole p-toluenesulfonic acid salt

5 ml of ethanol and 30 ml of water were added to 2.48 g of the crystal obtained in 4), and after 3.8 g of p-toluenesulfonic acid monohydrate was further added thereto, the mixture was stirred at room temperature for 5 hours. The precipitated crystal was filtered, subsequently washed with a mixture of ethanol and water (1:6), water and then ethanol, and dried with a vacuum pump to yield 3.5 g of white crystal.

¹H-NMR (DMSO-d₆) δppm: 2.28 (3H, s), 7.12 (2H, dd, J=7.75 & 0.50 Hz), 7.48 (2H, dd, J=7.75 & 0.50 Hz), 8.33 (1H, dd, J=5.12 & 1.65 Hz), 8.45 (2H, d, J=6.11 Hz), 8.57 (1H, dd, J=1.65 & 0.66 Hz), 8.96˜9.02 (3H, m)

6) Production of the object compound

17 ml of ethanol and 17 ml of water were added to 3.36 g of the crystal obtained in 5), and the mixture was stirred at room temperature for 30 minutes. A solution of sodium carbonate (0.74 g of sodium carbonate in 17 ml of water) was further added, and the mixture was stirred at room temperature for 2 hours. The precipitated crystal was filtered, subsequently washed with water and ethanol, and dried with a vacuum pump to yield 1.89 g of the object compound as a pale yellow crystal.

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TOPIROXOSTAT

SYNTHESIS

WO2014017516A1

(First step)
The first step, 4 – is a step of obtaining a compound (3) is reacted in the presence of an alkali metal alkoxide, cyano-N-oxide and (2), and isonicotinic acid hydrazide.

4 used in this reaction – isonicotinic acid hydrazide and (2) a cyano-N-oxide is a known compound both, I can be prepared by known means.
The alkali metal alkoxide is used, ₆ alkoxide alkali metal C _1-C are preferred, sodium methylate, sodium ethylate and the like can be given as specific examples. The reaction is preferably carried out in a solvent, as the solvent, alcohol solvents such as methanol, ethanol and the like are preferable.

The reaction is preferably first in a solvent, is treated with an alkali metal alkoxide compound (2) and then to react the isonicotinic acid hydrazide. First, heated to reflux under cooling, at 80 ℃ from 15 ℃ preferably, 30 minutes and 12 hours in general, the reaction temperature in the reaction with an alkali metal alkoxide (2) with the compound is reacted 1-4 hours, preferably about. Under the temperature conditions, using an excess amount or one equivalent of 30 minutes to 12 hours usually, reaction with isonicotinic acid hydrazide Subsequent to reaction for 1 to 5 hours, preferably.

Example 1:

Synthesis 4 oxide (3) – - – (4 – pyridin-carbonyl) -4 – N “pyridine hydrazide imide -1 was suspended in 40mL of methanol cyanopyridine-N-oxide and (2) 5.00g, sodium was added to methylate 22.4mg, and the mixture was stirred for 2 hours under 40 ℃ nitrogen atmosphere. was cooled to room temperature. reaction solution was stirred for 4 hours at 40 ℃ was added isonicotinic acid hydrazide 5.71g at the same temperature, precipitated The filtrated crystals were, washed with methanol 15mL, and dried 15 hours at 80 ℃, N “- to give (3) 9.60g oxide – (4 – pyridin) -4 – pyridine-hydrazide imide -1.
¹ H-NMR (DMSO-d ₆₎ ^δ (ppm): 6.98 (br, 2H), 7.81 (d, 2H, J = 5.77Hz), 7.85 (d, 2H, J = 7 .09 Hz), 8.29 (d, 2H, J = 7.09Hz), 8.73 (d, 2H, J = 5.77Hz), 10.37 (br, 1H)
MS m / z: 256 [M-H] ^-

(Second step)
The second step is a step of obtaining compound (4) by cyanation agent cyano compound (3).

As the cyanation agent used, trialkyl cyanide alkali metal cyanide, sodium cyanide, potassium cyanide and the like, zinc cyanide, trimethylsilyl cyanide and the like.

The cyanation reaction is preferably, for example, be carried out (Heterocycles, Vol.22, No.5, 1994) by Reissert Henze reaction. This reaction, for example, to give compound (4) by an organic solvent in the compound (3), and after activation with carbamoyl halide, and reacting the cyano agent. The alkylcarbamoyl halide used in the carbamoylation is a first step in Reissert Henze reaction, ₆ alkylcarbamoyl halide di C _1-C dimethylcarbamoyl chloride, and di-propyl carbamoyl chloride can be used, preferably, dimethylcarbamoyl is chloride. The solvent used in this reaction, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, tetrahydrofuran and acetonitrile can be used, however, N, N-dimethylformamide is preferred. Further, 15 ~ 60 ℃, more preferably 30 ~ 50 ℃ reaction temperature. The reaction time is preferably 1 to 24 hours, more preferably 1 to 3 hours. As the cyanation agent used in the cyanation reaction followed, cyano agents above can be used, sodium cyanide, potassium cyanide, zinc cyanide, and trimethylsilyl cyanide, and more preferably, it is sodium cyanide . -20 ~ 60 ℃ is preferred, more preferably -10 ~ 40 ℃, reaction temperature is 1-4 hours.

Is a novel compound (4) The compound obtained in this second step, it is useful as an intermediate for the production of compound (1). If through Compound (4) can be synthesized in good yield and easily without the need for purification in the second step is also possible, and can be produced (1) Compound industrially efficiently compound (4).

Synthetic N “hydrazide (4) – (4 – pyridine carbonyl) -4 – pyridine carboxylic acid N’-(carboxylic imidoyloxy – 2 – - cyano-4)

Example 2

4 pyridine hydrazide imide -1 – oxide ( was suspended in N, N-dimethylformamide 48mL and 3) 10.0g, under nitrogen atmosphere, followed by stirring for 1 hour was added dimethylcarbamoyl chloride 9.20g at 40 ℃. was added sodium cyanide 2.48g at the same temperature, After cooling to 5 ℃ below. reaction mixture was stirred for 1 hour, the crystals were collected by filtration. precipitate was successively added dropwise a 5% aqueous sodium bicarbonate solution 100mL, and 100mL water, and washed with water 100mL, at 80 ℃ for 15 h and dried under reduced pressure to give 4 – hydrazide (4) 9.28g of pyridine-carboxylic acid N’-(carboxylic imide yl – 2 – cyano-4).
¹ H-NMR (DMSO-d ₆₎ ^δ (ppm): 7.15 (br, 2H), 7.82 (d, 2H, J = 5.61Hz), 8.14 (d, 1H, J = 5 .11 Hz), 8.37 (s, 1H), 8.75 (d, 2H, J = 5.61Hz), 8.86 (d, 1H, J = 5.11Hz), 10.47 (br, 1H )
MS m / z: 265 [M-H] ^-

(Third step)
The third step is a step of obtaining a compound (1) by the presence of an acid catalyst, the cyclization reaction of the compound (4).

As the acid, organic phosphoric acid, p-toluenesulfonic acid, such as hydrochloric acid, inorganic acids can be used, inorganic acids, phosphoric acid is particularly preferable. As the reaction solvent, water, 2 – butanol, 2 – mixed solvent of alcohol and water or alcohol, propanol, ethanol and the like can be used, but water and 2 – I was mixed 5:1 to 10:1 butanol solvent. The reaction temperature and time, 60 ~ 100 ℃, preferably 2 to 12 hours at 70 ~ 90 ℃, I want to 8-10 hours, preferably.

Intermediates and compounds of the present invention the method (1) can be isolated and purified from the washed reaction mixture, recrystallization, by means of various conventional chromatography.

Example 3:

4 – [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazol-3 - yl] pyridine-2 – carbonitrile 4 Synthesis of (1) – pyridine-carboxylic acid N’- (2 – cyano-4 – carboxylic imide yl) water 82mL, 2 hydrazide (4) 9.25g – butanol was added 8.2mL, phosphate 4.00g, was stirred for 8 h at 80 ℃. After cooling to room temperature, the reaction mixture was precipitated crystals were collected by filtration, water: 2 – were washed with a mixed solution of 92.5mL butanol = 10:1. The 13 h and dried under reduced pressure at 80 ℃ crystals obtained 4 – [5 - (pyridin-4 - yl) - ^{1 H-1,} 2,4 - triazol-3 - yl] pyridine-2 – carbonitrile (1 I got a) 7.89g.

Topiroxostat

¹ H-NMR (DMSO-d ₆₎ ^δ (ppm): 8.02 (dd, 2H, J = 4.59,1.62 Hz), 8.32 (dd, 1H, J = 5.13,1. 62Hz), 8.55 (dd, 1H, J = 1.62,1.08 Hz), 8.80 (dd, 2H, J = 4.59,1.62 Hz), 8.93 (dd, 1H, 5 .13,1.08 Hz)
MS m / z: 247 [M-H] ^-

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WO2014017515A1

Synthetic water-carbonitrile p-toluenesulfonate – pyridine Example 1: 4 – [yl 5 - (pyridin-4 - yl)-1H-1, 2,4 - - -3 triazol]: 2 – butanol = was added monohydrate 6.62g p-toluenesulfonic acid in a mixed solution of 55mL of 10:1, 4 at 80 ℃ – [5 - (pyridin-4 - yl)-1H-1, 2,4 - yl] pyridine-2 – - triazol-3 was added carbonitrile 7.85g, and the mixture was stirred at the same temperature for 1 hour. After cooling to room temperature, the reaction mixture, and the precipitated crystals were collected by filtration, and water: 2 – were washed with a mixed solution of 40mL of butanol = 10:1. The dried under reduced pressure for 10 hours at 80 ℃ crystals obtained 4 – [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazol-3 - yl] pyridine-2 – carbonitrile p-toluene I got a sulfonate 12.6g.
¹ H-NMR (DMSO-d ₆₎ ^δ (ppm): 2.29 (s, 3H), 7.11 (m, 2H), 7.48 (dd, 2H, J = 6.48,1.62 Hz ) ,8.32-8 .35 (m, 3H), 8.57 (dd, 1H, J = 1.62,0.81 Hz) ,8.94-8 .98 (m, 3H)

- [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazole and potassium carbonate 8.22g, 4 in a mixed solution of 80mL of ethanol = 9:1: preparation water of crystal form I: Example 2 I was dissolved carbonitrile p-toluenesulfonate 10.0g - -3 - yl] pyridine-2. After stirring for 5 hours plus 15mL 6M hydrochloric acid at 20 ℃, was the precipitated crystals were collected by filtration, and washed with water 100mL. The 23 h and dried under reduced pressure at 80 ℃, 4 – to obtain carbonitrile 5.78g – pyridin-2 [yl 5 - (pyridin-4 - yl)-1H-1, 2,4 - - -3 triazole. Having a DSC as shown in FIG 4 and the powder X-ray diffraction pattern shown in FIG 1, the resulting crystals were type-I crystals.
¹ H-NMR (DMSO-d ₆₎ ^δ (ppm): 8.02 (dd, 2H, J = 4.59,1.62 Hz), 8.32 (dd, 1H, J = 5.13,1. 62Hz), 8.55 (dd, 1H, J = 1.62,1.08 Hz), 8.80 (dd, 2H, J = 4.59,1.62 Hz), 8.93 (dd, 1H, 5 .13,1.08 Hz)
Melting point: 327 ℃

N, N carbonitrile 40.0g - preparation of 4 Form II - [5 - (pyridin-4 - yl)-1H-1, 2,4 - yl - triazol-3]-2: Example 3 – dimethylformamide was added 300mL, and stirred for 25 min at 150 ℃. After cooling to room temperature the solution, and the precipitated crystals were collected by filtration, and washed twice with water 200mL, 4 and dried under reduced pressure overnight at 80 ℃ the crystal – [5 - (pyridin-4 - yl)-1H-1 , 2,4 - I got carbonitrile 30.4g - yl] pyridine-2 – triazole-3. Having a DSC as shown in FIG 5 and powder X-ray diffraction pattern shown in FIG 2, the resulting crystals were type II crystals.
¹ H-NMR (DMSO-d ₆₎ ^δ (ppm): 8.02 (dd, 2H, J = 4.59,1.62 Hz), 8.32 (dd, 1H, J = 5.13,1. 62Hz), 8.55 (dd, 1H, J = 1.62,1.08 Hz), 8.80 (dd, 2H, J = 4.59,1.62 Hz), 8.93 (dd, 1H, 5 .13,1.08 Hz)
Melting point: 327 ℃

The 25 ℃, about 2g carbonitrile, – preparation of the hydrate 4 – [5 - (pyridin-4 - yl)-1H-1, 2,4 - triazol-3 - yl] pyridine-2: Example 4 I was stored for 14 days under conditions of relative humidity 97%. Having a DSC as shown in FIG 7 and the powder X-ray diffraction pattern shown in FIG 3, the obtained crystal was a hydrate.
¹ H-NMR (DMSO-d ₆₎ ^δ (ppm): 8.02 (dd, 2H, J = 4.59,1.62 Hz), 8.32 (dd, 1H, J = 5.13,1. 62Hz), 8.55 (dd, 1H, J = 1.62,1.08 Hz), 8.80 (dd, 2H, J = 4.59,1.62 Hz), 8.93 (dd, 1H, 5 .13,1.08 Hz)
Melting point: 327 ℃

Test Example: solubility test Type I crystal by crystal form, II-type crystal, and water solubility of the hydrate was calculated by absorbance measurement method, a saturated solution concentration of each sample. I Figure 8 shows the results.Whereas the 6.2μg/mL water solubility of crystalline Form I, II type crystal 4.2μg/mL, hydrate was 1.9μg/mL.
From Figure 8, the water solubility of Form II and Form I crystals is good, water-soluble type I crystal is particularly good.

……………….

NMR

BMCL Volume 19, Issue 21, 1 November 2009, Pages 6225–6229

http://www.sciencedirect.com/science/article/pii/S0960894X09012372?np=y

view compd 39 and ignore rest

TOPIROXOSTAT, FYX O51

view compd 39 and ignore rest

SUPP INFO…….https://docs.google.com/viewer?url=http://www.sciencedirect.com/science/MiamiMultiMediaURL/1-s2.0-S0960894X09012372/1-s2.0-S0960894X09012372-mmc1.doc/271398/FULL/S0960894X09012372/50d911fe734c16dfb94912d481cb466a/mmc1.doc

Nucleosides, Nucleotides and Nucleic Acids, 2008 ,  vol. 27,  6-7  pg. 888 – 893

Inoue, Tsutomu; Sato, Takahiro; Ashizawa, Naoki; Iwanaga, Takashi; Matsumoto, Koji; Nagata, Osamu; Nakamura, Hiroshi
Bioorganic and Medicinal Chemistry Letters, 2009 ,  vol. 19,   21  pg. 6225 – 6229

WO 2012060308

WO 2007148835

WO 2005009991

SUMATRIPTAN, GR-43175

1-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]- N-methyl-methanesulfonamide

3-[2-(Dimethylamino)ethyl]-N-methyl-1H-indole-5-methanesulfonamide

Avanir Pharmaceuticals has filed a new drug application (NDA) with the US Food and Drug Administration (FDA) for approval of its new breath-powered investigational drug-device combination product, ‘AVP-825′, for the acute treatment of migraines.  click on title  Avanir files new drug application for migraine drug 

SUMATRIPTAN

SUMATRIPTAN SUCCINATE

AVP-825 is an investigational drug-device combination product consisting of low-dose sumatriptan powder delivered intranasally utilizing a novel Breath Powered delivery technology. If approved, AVP-825 would be the first and only fast-acting, dry-powder intranasal form of sumatriptan for the treatment of migraine.

The Breath Powered delivery technology is activated by user’s breath to propel medications deep into the nasal cavity where absorption is more efficient and consistent than through most other routes. A user exhales into the device, automatically closing the soft palate and sealing off the nasal cavity completely. Through a sealing nosepiece placed into the nostril, the exhaled breath carries medication from the device directly into one side of the nose. Narrow nasal passages are gently expanded and medication is dispersed deep into the nasal cavity reaching areas where it can be rapidly absorbed. As the medication is delivered, the air flows around to the opposite side of the nasal cavity and exits through the other nostril. Closure of the soft palate helps prevent swallowing or inhalation of sumatriptan powder into the lungs.

Sumatriptan is a synthetic drug belonging to the triptan class, used for the treatment of migraine headaches. Structurally, it is an analog of the naturally occurring neuro-active alkaloids dimethyltryptamine (DMT), bufotenine, and 5-methoxy-dimethyltryptamine, with an N-methyl sulfonamidomethyl- group at position C-5 on the indole ring.^[1]

Sumatriptan is produced and marketed by various drug manufacturers with many different trade names such as Sumatriptan, Imitrex, Treximet, Imigran, Imigran recovery.

Large doses of sumatriptan can cause sulfhemoglobinemia, a rare condition in which the blood changes from red to greenish-black, due to the integration of sulfur into the hemoglobin molecule.^[2] If sumatriptan is discontinued, the condition reverses within a few weeks.

Serious cardiac events, including some that have been fatal, have occurred following the use of sumatriptan injection or tablets. Events reported have included coronary artery vasospasm, transient myocardial ischemia, myocardial infarction, ventricular tachycardia, and ventricular fibrillation.

The most common side-effects^[3] reported by at least 2% of patients in controlled trials of sumatriptan (25, 50, and 100 mg tablets) for migraine are atypical sensations (paresthesias and warm/cold sensations) reported by 4% in the placebo group and 5–6% in the sumatriptan groups, pain and other pressure sensations (including chest pain) reported by 4% in the placebo group and 6–8% in the sumatriptan groups, neurological events (vertigo) reported by less than 1% in the placebo group and less than 1% to 2% in the sumatriptan groups. Malaise/fatigue occurred in less than 1% of the placebo group and 2–3% of the sumatriptan groups. Sleep disturbance occurred in less than 1% in the placebo group to 2% in the sumatriptan group.

SUMATRIPTAN

Sumatriptan is structurally similar to serotonin (5HT), and is a 5-HT (types 5-HT_1D and 5-HT_1B^[4]) agonist. The specific receptor subtypes it activates are present on the cranial arteries and veins. Acting as an agonist at these receptors, sumatriptan reduces the vascular inflammation associated with migraines.

The specific receptor subtype it activates is present in the cranial and basilar arteries. Activation of these receptors causes vasoconstriction of those dilated arteries. Sumatriptan is also shown to decrease the activity of the trigeminal nerve, which, it is presumed, accounts for sumatriptan’s efficacy in treating cluster headaches. The injectable form of the drug has been shown to abort a cluster headache within fifteen minutes in 96% of cases.^[5]

Sumatriptan is administered in several forms; tablets, subcutaneous injection, and nasal spray. Oral administration (as succinate) suffers from poorbioavailability, partly due to presystemic metabolism—some of it gets broken down in the stomach and bloodstream before it reaches the target arteries. A new rapid-release tablet formulation has the same bioavailability, but the maximum concentration is achieved on average 10–15 minutes earlier. When injected, sumatriptan is faster-acting (usually within 10 minutes), but the effect lasts for a shorter time. Sumatriptan is metabolised primarily by monoamine oxidase A into an indole acetic acid analogue, part of which is further conjugated with glucuronic acid. These metabolites are excreted in the urine and bile. Only about 3% of the active drug may be recovered unchanged.

There is no simple, direct relationship between sumatriptan concentration (pharmacokinetics) per se in the blood and its anti-migraine effect (pharmacodynamics). This paradox has, to some extent, been resolved by comparing the rates of absorption of the various sumatriptan formulations, rather than the absolute amounts of drug that they deliver.^[6][7]

Sumatriptan was the first clinically available triptan (in 1991). In the United States, it is available only by medical prescription. However, it can be bought over the counter in the UK and Sweden in 50 mg dosage. Several dosage forms for sumatriptan have been approved, including tablets, solution for injection, and nasal inhalers.

On April 15, 2008, the US FDA approved Treximet, a combination of sumatriptan and naproxen, an NSAID.^[8] This combination has shown a benefit over either medicine used separately.^[9]

In July 2009, the US FDA approved a single-use jet injector formulation of sumatriptan. The device delivers a subcutaneous injection of 6 mg sumatriptan, without the use of a needle.Autoinjectors with needles have been previously available in Europe and North America for several years.^[10]

Phase III studies with a iontophoretic transdermal patch (Zelrix/Zecuity) started in July 2008.^[11] This patch uses low voltage controlled by a pre-programmed microchip to deliver a single dose of sumatriptan through the skin within 30 minutes.^[12][13]Zecuity was approved by the US FDA in January 2013.^[14]

Sumatriptan vials 100 5509

On November 6, 2008, Par Pharmaceutical announced that it would begin shipping generic versions of sumatriptan injection (sumatriptan succinate injection) 4 mg and 6 mg starter kits and 4 mg and 6 mg pre-filled syringe cartridges to the trade immediately. In addition, Par anticipates launching the 6 mg vials early in 2009.^[15]

Mylan Laboratories Inc., Ranbaxy, Sandoz, Dr. Reddy’s Pharmaceuticals and other companies have received FDA approval for generic versions of Imitrex tablets in 25-, 50-, and 100-milligram doses since 2009. The drug is available in U.S. and European markets, since Glaxo’s patent protections have expired in those jurisdictions. However, sales of a generic delivered via nasal spray are still restricted in the United States.

See also Sumavel DosePro (above).^[10]

Chemistry

hydrogenation of nitrile with pd/c in presence of dimethyl amine

…………………

U.S. Patent 4,785,016

The diazotation of 4-amino-N-methylbenzenemethanesulfonamide (I) with NaNO2-HCl followed by reduction with SnCl2 gives the 4-hydrazino compound (II), which is condensed with (phenylthio)acetaldehyde (III) in ethanol yielding the ethylideneamino compound (IV). The cyclization of (IV) with HCl in ethanol affords N-methyl-3-(phenylthio)-1H-indole-5-methansulfonamide (V), which is desulfurized with RaNi in refluxing ethanol-water to give N-methyl-1H-indole-5-methanesulfonamide (VI). The reaction of (VI) with oxalyl chloride and dimethylamine yields the oxalyl derivative (VII), which is finally reduced with LiAlH4 in refluxing THF.

The condensation of hydrazine (II) with 4,4-dimethoxy-N,N-dimethylbutylamine (VIII) by means of HCl in water gives the butylidenehydrazino compound (IX), which is cyclized with polyphosphate ester (PPE) in CHCl3.

……………………

Beilstein J. Org. Chem. 2011, 7, 442–495.

http://www.beilstein-journals.org/bjoc/single/articleFullText.htm?publicId=1860-5397-7-57#S9

ref are below article

Indoles

The neuroamine transmitter serotonin contains an indole ring, so it is not surprising that indoles are a recurring theme in many drugs affecting central nervous system (CNS) function including antidepressants, antipsychotics, anxiolytics and antimigraine drugs, as well as psychedelic agents. Indole is also one of the best represented heterocyclic motifs present in the top selling pharmaceuticals, being found in eight of the top 200 drugs, with five of these belonging to the triptan family of antimigraine treatments. The classical Fischer indole synthesis is usually reported as one of the first choice routes to prepare these scaffolds. Drugs such as GSK’s serotonin receptor modulators sumatriptan (49, Imitrex) and zolmitriptan (50, Zomig) use the Fischer indole synthesis at a late stage in order to form the desired compound albeit in only low to moderate yields (Scheme 9).

However, in sumatriptan the indole product resulting from the Fischer synthesis can still react further which leads to the formation of by-products and significantly reduced yields. One way to minimise this was to protect the nitrogen of the sulfonamide group prior to indole formation [11]. This leads not only to an increased yield in the indole forming step (to 50%) but also facilitates chromatographic purification. The dimethylamino group can be present from the beginning of the synthesis or can be introduced via displacement of chloride or reduction of a cyano moiety. Alternatively, the dimethyl ethylene amine side chain can be introduced in position 3 via a Friedel–Crafts-type acylation. The resulting acid chloride is transformed in situ to the corresponding amide which on reduction with lithium aluminium hydride affords sumatriptan (Scheme 10) [12].

In the standard Fischer indole synthesis a hydrazine, which is most commonly derived from the corresponding diazonium salt, is reacted with a suitable carbonyl compound. Alternatively, the Japp–Klingemann reaction can be used to directly couple the diazonium salt with a β-ketoester to obtain a hydrazone which can then undergo indole ring formation (Scheme 11) [13].

As can be seen from Scheme 11 the indole 59 prepared via the Japp–Klingemann reaction is substituted at position 2 by an ester group which prevents reaction with electrophiles, thereby reducing the amount of undesired by-products. A simple sequence of hydrolysis and decarboxylation then affords sumatriptan [14].

All the reported methods for the synthesis of sumatriptan begin with the sulfonamide group already present on the aromatic ring and several routes are possible to introduce this functional group. The scalable route to the sulfonamides inevitably involves the preparation of the sulfonyl chloride intermediate which is then trapped with the desired amine. The sulfonyl chloride can also be prepared from the corresponding hemithioacetal 61 by treatment with NCS in wet acetic acid (Scheme 12). This efficient oxidation produces only methanol and formaldehyde as by-products [15].

Scheme 12: Synthesis of the intermediate sulfonyl chlorides 62 and 63.

1. 11. Pete, B.; Bitter, I.; Szántay, C., Jr.; Schön, I.; Töke, L. Heterocycles 1998, 48, 1139–1149. doi:10.3987/COM-97-8087
2. 12…Oxford, A. W. Indole Derivative. U.S. Patent 5,037,845, Aug 6, 1991.
3. 13…Japp, F. R.; Klingemann, F. Chem. Ber. 1887, 20, 2942–2944. doi:10.1002/cber.188702002165
4. Pete, B.; Bitter, I.; Harsányi, K.; Töke, L. Heterocycles 2000, 53, 665–673. doi:10.3987/COM-99-8815
5. Kim, D.-W.; Ko, Y. K.; Kim, S. H. Synthesis 1992, 12, 1203–1204. doi:10.1055/s-1992-26333
   [15

References for full article

Avanir Pharmaceuticals, Inc. is a biopharmaceutical company focused on bringing innovative medicines to patients with central nervous system disorders of high unmet medical need. As part of our commitment, we have extensively invested in our pipeline and are dedicated to advancing medicines that can substantially improve the lives of patients and their loved ones. For more information about Avanir, please visit http://www.avanir.com.

AVANIR® is a trademark or registered trademark of Avanir Pharmaceuticals, Inc. in the United States and other countries. All other trademarks are the property of their respective owners.

Avanir Pharmaceuticals, Inc. licensed exclusive rights for the development and commercialization of AVP-825, a novel Breath Powered intranasal system containing a low-dose sumatriptan powder from OptiNose Inc. of Yardley, PA.

IMITREX Tablets contain sumatriptan succinate, a selective 5-HT1B/1D receptor agonist. Sumatriptan succinate is chemically designated as 3-[2-(dimethylamino)ethyl]-N-methyl-indole- 5-methanesulfonamide succinate (1:1), and it has the following structure:

IMITREX Tablets contain sumatriptan succinate, a selective 5-HT_1B/1Dreceptor agonist. Sumatriptan succinate is chemically designated as 3-[2-(dimethylamino)ethyl]-N-methyl-indole- 5-methanesulfonamide succinate (1:1), and it has the following structure:

The empirical formula is C₁₄H₂₁N₃O₂S•C₄H₆O₄, representing a molecular weight of 413.5. Sumatriptan succinate is a white to off-white powder that is readily soluble in water and in saline.

Each IMITREX Tablet for oral administration contains 35, 70, or 140 mg of sumatriptan succinate equivalent to 25, 50, or 100 mg of sumatriptan, respectively. Each tablet also contains the inactive ingredients croscarmellose sodium, dibasic calcium phosphate, magnesium stearate, microcrystalline cellulose, and sodium bicarbonate. Each 100-mg tablet also contains hypromellose, iron oxide, titanium dioxide, and triacetin.

WO2008067465A1 OR US8084605  IS THE PATENT USED AND WITH FORM “A” AND AMORPHOUS FORM

Active pharmaceutical ingredients (APIs), frequently delivered to the patient in the solid-state as part of an approved dosage form, can exist in such diverse solid forms as polymorphs, pseudopolymorphs, salts, co-crystals and amorphous solids. Various solid forms often display different mechanical, thermal, physical and chemical properties that can remarkably influence the bioavailability, hygroscopicity, stability and other performance characteristics of the drug.

Hence, a thorough understanding of the relationship between the particular solid form of an active pharmaceutical ingredient (API) and its functional properties is important in selecting the most suitable form of the API for development into a drug product. In past decades, there have been significant efforts on the discovery, selection and control of the solid forms of APIs and bulk drugs.

If you’re involved in late drug discovery, API manufacture, drug product formulation, clinical material production, or manufacture of final dosage form, a basic understanding and awareness of solid form issues could save you a great deal of difficulty, time, and money during drug development.

 

What is polymorphism?

Polymorphs are crystalline materials that have the same chemical composition but different molecular packing. The concept is well demonstrated by the different crystalline forms of carbon. Diamond, graphite, and fullerenes are all made of pure carbon, but their physical and chemical properties vary drastically. Polymorphs are one type of solid form. Other solid form types include solvates, hydrates, and amorphous forms.

Solvates are crystalline materials made of the same chemical substance, but with molecules of solvent regularly incorporated into a unique molecular packing. When water is the solvent, these are called hydrates. An amorphous form of a substance has the same chemical composition, but lacks the long-range molecular order of a crystalline form of the same substance. Many organic and inorganic compounds, including APIs, can exist in multiple solid forms.

Some APIs may have only one or two known solid forms. Others may exist in twenty different forms, each having different physical and chemical properties.

Solid form screening, including salt, polymorph, cocrystal and amorphous solid dispersions, is vitial for successful pharmaceutical development. With an increase in the size and complexity of the molecules that enter into drug development, companies face a larger number of compounds that are either poorly soluble, difficult to crystallize or problematic with respect to desired physical chemical properties hindering successful drug development.

Crystallics has an extensive track record in executing solid state research studies and its research team has a broad expertise in identifying new crystal forms as well as in solving problems related to polymorphism and crystallization.

Investigational new drug, writing an application for clinical trial authorization, permission marketing …The control of polymorphism in drug candidates is now ubiquitous.

READ………….An Overview of Solid Form Screening During Drug Development, http://www.icdd.com/ppxrd/10/presentations/PPXRD-10_Ann_Newman.pdf

ANN NEWMAN

When addressing the subject of polymorphism, the first reference that comes to mind is that of the occurred during the manufacture of ritonavir incident. Abbott molecule inhibitor of HIV protease marketed as Norvir, is a cautionary example of the challenges of polymorphism. 

Indeed, during the production of ritonavir in 1997, a new polymorph unmarked emerged. Its precipitation and unexpected outbreak led to the cessation of the production of Norvir and seriously compromise the process. The incident has deeply marked the pharmaceutical industry.

It is ironic that the process used to discover pharmaceutical drug targets is the same one that decreases the actual efficacy of those drugs once ingested. If you remember from basic chemistry, there are compounds that exist in highly ordered crystalline states and those that remain in amorphous form.

The discovery of drug targets has often been accomplished through X-ray crystallography, which requires a sample (for example, of a defective enzyme linked to cancer or high cholesterol) to be crystallized so that the diffraction patterns can be made sense of. Scientists may spend years trying to crystallize one molecule or compound so that they can identify regions that, for example, may be blocked by pharmaceuticals.

However, when it comes to the molecular arrangement of those pharmaceuticals, crystallization actually decreases their bioavailability and solubility. Thus, it may be better for these drugs to be in amorphous form. Pierric Marchand, general manager of the company Holodiag, dedicated to the study and characterization of solid state, summarizes that “ today, it is not reasonable to not worry about the problem of polymorphism ” .

“ In recent years, manufacturers have realized the essential side of expertise , “says Jean-Rémi David, commercial director Calytherm. The services company specializing in the field of physico-chemical analysis, based in Herault, has just relocated last year in supporting pharmaceutical development to meet demand. ” This is a concern for all deal with potential impacts on the effectiveness or the formulation , “says Stéphane Suchet, quality manager in the group of fine and specialty chemicals Axyntis.

Polymorphic forms are the amorphous and crystalline forms such as hydrate or solvate forms. When a molecule of interest exists in polymorphic forms, it is called polymorphism, according to the definition of the FDA (Food and Drug Administration).

Polymorphism is present at all stages of development of a drug from research to marketing. ” Keep in mind that organic molecule loves to polymorphism , “says Marchand Pierric. However, for a marketing authorization for example, must learn the criteria for the polymorphism of the molecule. ” In terms of the formulation, for example we can check whether the selected polymorphism is unchanged , “explains Pierric Marchand.

A significant influence on several levels  Because the consequences of polymorphism are multiple. ” They are at three levels: bioequivalence, manufacturability and stability ”lists Fabienne Lacoulonche, founder and scientific director of Calytherm.In terms of bioequivalence, different polymorphic forms may have different properties of solubility and dissolution rate … ” For poorly soluble active ingredients, you can have much more bioavailable than other crystalline forms , “Fabienne Lacoulonche information.

In terms of manufacturability, some parameters such as temperature, moisture can lead to changes in the crystalline form. ” The complexity is to anticipate changes polymorphism, both at laboratory scale, pilot and industrial , “adds the founder of Calytherm. Finally, polymorphism plays on stability. Active ingredient or finished product, are subjected to stability studies in this direction. ” When the molecule is identified, we try to highlight the existence of several forms of polymorphism, explains Stéphane Suchet (Axyntis) . 

When developing a new substance, the assessment is systematic . “ Isolation of crystals from a screening is carried out in different solvents by various analytical techniques. Ideally, it will be concluded the absence of polymorphism. ” But if different polymorphic forms are present, we rework the terms of our crystallization process to control the formation of the same polymorph reproducibly ideally form the thermodynamically more stable , “says Stéphane Suchet. X-ray diffraction and other thermal analysis “

The ICH guidelines provide decision trees to guide the industry in controlling polymorphism says Fabienne Lacoulonche (Calytherm.) We use it for writing the CTD (Common Technical Document) .

“Polymorphism is a phenomenon” complex and difficult to control, because the crystallization is dependent on many parameters , she develops. must understand the maximum . “ For this, several analytical methods are available to industry. The main technique is the X-ray diffraction ” It is a robust, rapid, which allows to characterize the different polymorphs , “summarizes Pierric Marchand (Holodiag).Non-destructive, it can work both on small quantities on large samples. Temperature and atmosphere are controlled, and analytical capabilities are broad.

But if this technique indispensable allows for routine and development, it is not sufficient in itself. Just to add a battery of additional tests, thermal analysis. ” It takes coupling methods “ confirms Fabienne Lacoulonche (Calytherm). The X-ray diffraction is a method of choice, but sometimes it is not sufficient.

The coupling with a thermal analysis method (technical ATG, or DSC thermal analysis, differential scanning calorimetry or thermomicroscopique) allows to distinguish between two polymorphic whose RX diffractograms obtained are comparable.

TGA can be coupled with IR or mass spectrometry, DSC with RX. Raman spectroscopy is also part of complementary methods. ” The difficulty increases when we want to characterize the shape of the active ingredient in the finished product , says Fabienne Lacoulonche. example, by X-ray diffraction, the peaks related to the active ingredient in the diffractogram of the finished product may be masked by those excipients: it is then necessary to use other methods, such as Raman microscopy. ”In general, a single method of analysis is not sufficient to characterize the polymorphism of an active substance in the active substance or finished product: the complementarity of different methods that will conclude precisely on the polymorphism of a crystalline substance.

In addition, “ the diffractometer remains an expensive device, which requires installation in an air-conditioned and a cooling room , “says Marchand Pierric (Holodiag). To this is added the need to have expertise and qualified personnel to carry out the analyzes. ” We must master these techniques and the ability to interpret the results , “says Jean-Rémi David (Calytherm). However, polymorphism is a “problem well under control , “said Stéphane Suchet (Axyntis),” systematically evaluated although it is however not always immune to miss a polymorphic form, knowing that the screening performed in the development can never be completely comprehensive … ”

FDA

FDA may refuse to approve an ANDA referencing a listed drug if the application contains insufficient information to show that the drug substance is the “same” as that of the reference listed drug. A drug substance in a generic drug product is generally considered to be the same as the drug substance in the reference listed drug if it meets the same standards for identity.

In most cases, the standards for identity are described in the USPalthough FDA may prescribe additional standards when necessary. Because drug product performance depends on the product formulation, the drug substance in a proposed generic drug product need not have the same physical form (particle size, shape, or polymorph form) as the drug substance in the reference listed drug. An ANDA applicant is required to demonstrate that the proposed product meets the standards for identity, exhibits sufficient stability and is bioequivalent to the reference listed drug.

FDA PRESENTATION……polymorphs and co-crystals – ICDD      Regulatory Considerations on Pharmaceutical Solids: Polymorphs/Salts and Co-Crystals.. THIS IS A MUST READ ITEM

Over the years FDA has approved many generic drug products based upon a drug substance with different physical form from that of the drug substance in the respective reference listed drug (e.g., warfarin sodium, famotidine, and ranitidine). Also many ANDAs have been approved in which the drug substances differed from those in the corresponding reference listed drugs with respect to solvation or hydration state (e.g., terazosin hydrochloride, ampicillin, and cefadroxil). Several regulatory documents and literature reports (67-69) address issues relevant to the regulation of polymorphism.

The concepts and principles outlined in these are applicable for an ANDA. However, certain additional considerations may be applicable in case of ANDAs. Often at the time FDA receives an ANDA a monograph defining certain key attributes of the drug substance and drug product may be available in the Unites States Pharmacopoeia (USP). These public standards play a significant role in the ANDA regulatory review process and in case of polymorphism, when some differences are noted, lead to additional requirements and considerations.

This commentary is intended to provide a perspective on polymorphism in pharmaceutical solid in the context of ANDAs. It highlights major considerations for monitoring and controlling drug substance polymorphs and describes a framework for regulatory decisions regarding drug substance “sameness” considering the role and impact of polymorphism in pharmaceutical solids.

Since polymorphs exhibit certain differences in physical (e.g., powder flow and compactability, apparent solubility and dissolution rate) and solid state chemistry (reactivity) attributes that relate to stability and bioavailability it is essential that the product development and the FDA review process pay close attention to this issue.

This scrutiny is essential to ensure that polymorphic differences (when present) are addressed via design and control of formulation and process conditions to physical and chemical stability of the product over the intended shelf-life, and bioavailability/bioequivalence.

Most pharmaceuticals are distributed as solid doseages. In order to take action, they must dissolve in the gut and be absorbed into the blood stream. In many cases, the rate at which the drug dissolves can limit its effectiveness. Pharmaceutical compounds can be packed into more than one arrangement in the solid states known as polymorphs. Rapid and efficient methods of polymorph formation can be used to increase drug efficacy and shelf life.

Regulatory agencies worldwide require that, as part of any significant filing, a company has to demonstrate that it has made a reasonable effort to identify the polymorphs of their drug substance and has checked for polymorph interconversions. Due to the unpredictable behaviour of polymorphs and their respective differences in physicochemical properties, companies also have to demonstrate consistency in manufacturing between batches of the same product. Proper understanding of the polymorph landscape and nature of the polymorphs will contribute to manufacturing consistency.

POLYMORPHISM AND PATENTS http://www.collegio.unibo.it/uploads/ideas/joelbernstein.pdf  A MUST CLICK FOR PHARMA CHEMISTS

READ………..High-throughput crystallization: polymorphs, salts, co-crystalsand solvates of pharmaceutical solidshttp://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.85.5397&rep=rep1&type=pdf

Definitions

“Crystalline”, as the term is used herein, refers to a material, which may be hydrated and/or solvated, and has sufficient ordering of the chemical moiety to exhibit a discernable diffraction pattern by XRPD or other diffraction techniques. Often, a crystalline material that is obtained by direct crystallization of a compound dissolved in a solution or by interconversion of crystals obtained under different crystallization conditions, will have crystals that contain the solvent used in the crystallization, termed a crystalline solvate. Also, the specific solvent system and physical embodiment in which the crystallization is performed, collectively termed crystallization conditions, may result in the crystalline material having physical and chemical properties that are unique to the crystallization conditions, generally due to the orientation of the chemical moieties of the compound with respect to each other within the crystal and/or the predominance of a specific polymorphic form of the compound in the crystalline material.

Depending upon the polymorphic form(s) of the compound that are present in a composition, various amounts of the compound in an amorphous solid state may also be present, either as a side product of the initial crystallization, and/or a product of degradation of the crystals comprising the crystalline material. Thus, crystalline, as the term is used herein, contemplates that the composition may include amorphous content; the presence of the crystalline material among the amorphous material being detectably among other methods by the composition having a discernable diffraction pattern.

The amorphous content of a crystalline material may be increased by grinding or pulverizing the material, which is evidenced by broadening of diffraction and other spectral lines relative to the crystalline material prior to grinding. Sufficient grinding and/or pulverizing may broaden the lines relative to the crystalline material prior to grinding to the extent that the XRPD or other crystal specific spectrum may become undiscernable, making the material substantially amorphous or quasi-amorphous. Continued grinding would be expected to increase the amorphous content and further broaden the XRPD pattern with the limit of the XRPD pattern being so broadened that it can no longer be discerned above noise. When the XRPD pattern is broadened to the limit of being indiscernible, the material may be considered no longer a crystalline material, but instead be wholly amorphous. For material having increased amorphous content and wholly amorphous material, no peaks should be observed that would indicate grinding produces another form.

“Amorphous“, as the term is used herein, refers to a composition comprising a compound that contains too little crystalline content of the compound to yield a discernable pattern by XRPD or other diffraction techniques. Glassy materials are a type of amorphous material. Glassy materials do not have a true crystal lattice, and technically resembling very viscous non-crystalline liquids. Rather than being true solids, glasses may better be described as quasi-solid amorphous material. “Broad” or “broadened”, as the term is used herein to describe spectral lines, including XRPD, NMR and IR spectroscopy, and Raman spectroscopy lines, is a relative term that relates to the line width of a baseline spectrum. The baseline spectrum is often that of an unmanipulated crystalline form of a specific compound as obtained directly from a given set of physical and chemical conditions, including solvent composition and properties such as temperature and pressure.

For example, broadened can be used to describe the spectral lines of a XRPD spectrum of ground or pulverized material comprising a crystalline compound relative to the material prior to grinding. In materials where the constituent molecules, ions or atoms, as solvated or hydrated, are not tumbling rapidly, line broadening is indicative of increased randomness in the orientation of the chemical moieties of the compound, thus indicative of an increased amorphous content. When comparisons are made between crystalline materials obtained via different crystallization conditions, broader spectral lines indicate that the material producing the relatively broader spectral lines has a higher level of amorphous material.

“About” as the term is used herein, refers to an estimate that the actual value falls within ±5% of the value cited. “Forked” as the term is used herein to describe DSC endotherms and exotherms, refers to overlapping endotherms or exotherms having distinguishable peak positions

.

Classes of multicomponent pharmaceutical materials. (a) Schematic of crystalline materials showing neutral and charged species. The red box indicates polymorphs are possible for all the multicomponent crystals contained within the box (adapted from Reference 7). (b) Schematic of amorphous solid dispersions showing binary, ternary, and quaternary possibilities for polymers and surfactants. Other solubilization techniques using cyclodextrins and phospholipids are included for completeness but have a different mechanism for solubilization when compared to polymer and surfactant systems.

The red box indicates that properties can change with water or solvent content. General methods for precipitating and crystallizing a compound may be applied to prepare the various polymorphs described herein. These general methods are known to those skilled in the art of synthetic organic chemistry and pharmaceutical formulation, and are described, for example, by J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure ” 4^th Ed. (New York: Wiley-Interscience, 1992).

In general, a given polymorph of a compound may be obtained by direct crystallization of the compound or by crystallization of the compound followed by inter-conversion from another polymorphic form or from an amorphous form. Depending on the method by which a compound is crystallized, the resulting composition may contain different amounts of the compound in crystalline form as opposed to as an amorphous material.

Also, the resulting composition may contain differing mixtures of different polymorphic forms of the compound. Compositions comprising a higher percentage of crystalline content {e.g., forming crystals having fewer lattice defects and proportionately less glassy material) are generally prepared when using conditions that favor slower crystal formation, including slow solvent evaporation and those affecting kinetics.

Crystallization conditions may be appropriately adjusted to obtain higher quality crystalline material as necessary. Thus, for example, if poor crystals are formed under an initial set of crystallization conditions, the solvent temperature may be reduced and ambient pressure above the solution may be increased relative to the initial set of crystallization conditions in order to slow down crystallization. Precipitation of a compound from solution, often affected by rapid evaporation of solvent, is known to favor the compound forming an amorphous solid as opposed to crystals. A compound in an amorphous state may be produced by rapidly evaporating solvent from a solvated compound, or by grinding, pulverizing or otherwise physically pressurizing or abrading the compound while in a crystalline state.

Seven crystalline forms and one amorphous solid were identified by conducting a polymorph screen (Example 3). Described herein are Form A, Form B, Form C, Form D, Form E, Form F, Form G, and Amorphous Form of Compound I. Where possible, the results of each test for each different polymorph are provided. Forms A, C, D and E were prepared as pure forms. Forms B, F, and G were prepared as mixtures with Form A.
Various tests were performed in order to physically characterize the polymorphs of Compound I including X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), hot stage microscopy, Fourier transform infrared spectroscopy (FT-IR), Fourier transform Raman spectrometry, linked thermogravimetric-infrared spectroscopy (TG-IR), solution proton nuclear magnetic resonance (¹H-NMR), solid state ¹³carbon nuclear magnetic resonance (¹³C-NMR), and moisture sorption and desorption analysis (M S/Des).

Salt screening

Physicochemical properties of drug substances, such as solubility, dissolution rate, and physicochemical stability can be altered significantly by salt formation. Consequently, important properties of the drug product such as bioavailability or shelve life can be radically influenced. Crystallics’ technology platform for crystallization screening accommodates salt screening studies using only minimal amounts of drug substance while still performing a large number of experiments. High-throughput salt screening is used for both early phase salt selection studies and broad patent protection.

Salt selection – A powerful strategy for crystal form optimization

Pharmaceutical developers have focused efforts on finding and formulating a thermodynamically stable crystalline form with acceptable physical properties for a given compound. This is reasonable, given the need to avoid cascading from a meta-stable form to a more stable one in unpredictable fashion.

Occasionally certain physical properties, such as low aqueous solubility, are limiting to performance of the compound, leading to poor oral bioavailability or insufficient solubility for an injection formulation. One of the main strategies used to affect physical performance of a compound and one that is often employed by pharmaceutical scientists is the practice of salt selection (23). At least half of compounds in marketed products are in the form of a salt for one reason or another.

This fact alone speaks to the versatility of the salt selection approach. Salt forms of a pharmaceutical can have many benefits, such as improved stability characteristics, optimal bioavailability and aqueous solubility for an injectable formulation. Salts, like all other crystalline forms, are subject to polymorphism and solvate formation, thus requiring the same form identification studies as are needed for a neutral compound.

A remarkable example of co-optimization of properties is indinavir (HIV protease inhibitor), which is marketed as the sulfate salt ethanol solvate (24,25) The crystalline free base has variable oral bioavailability in dogs (26,27) and humans (28). While acidic solutions of the base compound showed good oral pharmacokinetics, the stability of the drug in acidic solution is not consistent with a product (26). Therefore, the discovery of the salt form ensured both shelf stability and robust bioavailability performance. The salt selection strategy is limited in two ways.

First, salt formation relies on the presence of one or more ionizable functional groups in the molecule; many drugs and development compounds lack this feature.

Second, our ability to predict a priori whether a given compound will form a crystalline salt (or salts) is non-existent. The ability to actively identify crystalline salt forms has been confined to manual empirical evaluation using multiple salt formers for a given acid or base. Recently advances have been made in the area of high-throughput salt selection and crystal engineering strategies associated with salt formation (14,29-32).

In one case, we have advocated the simultaneous assessment of polymorphism as a way to help rank the developability of different crystalline salts (14). While salt forms will continue to have a prominent place in pharmaceutical science, the need for enhanced productivity dictates that every advantage must be sought to aid the design of an appropriate crystalline form of an active molecule.

Specifically, the ability to design scaffolds into crystalline forms will enhance our capacity to convert interesting molecules into effective drugs. Crystal engineering offers some additional tools in this regard.

CASE STUDY FORM A ONLY US8084605

TRELAGLIPTIN SUCCINATE

Form A may be prepared by crystallization from the various solvents and under the various crystallization conditions used during the polymorph screen (e.g., fast and slow evaporation, cooling of saturated solutions, slurries, and solvent/antisolvent additions). Tables B and C of Example 3 summarize the procedures by which Form A was prepared.

For example, Form A was obtained by room temperature slurry of an excess amount of Compound I in acetone, acetonitrile, dichloromethane, 1,4-dioxane, diethyl ether, hexane, methanol, isopropanol, water, ethylacetate, tetrahydrofuran, toluene, or other like solvents on a rotating wheel for approximately 5 or 7 days.

The solids were collected by vacuum filtration, and air dried in the hood. Also, Form A was precipitated from a methanol solution of Compound I by slow evaporation (SE). Form A was characterized by XRPD, TGA, hot stage microscopy, IR, Raman spectroscopy, solution 1H-NMR, and solid state 13C-NMR. Figure 1 shows a characteristic XRPD spectrum (CuKa, λ=1.5418A) of Form A. The XRPD pattern confirmed that Form A was crystalline. Major X-Ray diffraction lines expressed in °2Θ and their relative intensities are summarized in Table 1. Table 1. Characteristic XRPD Peaks (CuKa) of Form A

The above set of XRPD peak positions or a subset thereof can be used to identify Form A. One subset comprises peaks at about 11.31, 11.91, 12.86, 14.54, 15.81, 16.83, 17.59, 19.26, 19.52, 21.04, 22.32, 26.63, and 29.87 °2Θ. Another subset comprises peaks at about 11.31, 11.91, 19.26, 21.04, and 22.32 °2Θ; the peaks of this subset show no shoulder peaks or peak split greater than 0.2 °2Θ. Another subset comprises peaks at about 11.31, 11.91 and 22.32 °2Θ. Figure 2 is a TGA thermogram of Form A. TGA analysis showed that Form A exhibited insignificant weight loss when heated from 25 0C to 165 0C; this result is indicative that Form A was an anhydrous solid. Figure 3 shows a characteristic DSC thermogram of Form A. DSC analysis showed a single endothermic event occurred at approximately 195 0C (peak maximum). This endothermic event was confirmed by hot stage microscopy which showed the melting of Form A, which onset around 177 0C and the melting point estimated to be at approximately 184 0C.

US8084605

Figure 4 (A-D) shows a characteristic FT-IR spectrum of Form A. The major bands expressed in reciprocal wavelengths (wavenumber in cm”1) are positioned at about 3815, 3736, 3675, 3460, 3402, 3141, 3098, 3068, 3049, 2953, 2934, 2854, 2760, 2625, 2536, 2481, 2266, 2225, 2176, 1990, 1890, 1699, 1657, 1638, 1626, 1609, 1586, 1553, 1517, 1492, 1478, 1450, 1419, 1409, 1380, 1351, 1327, 1289, 1271, 1236, 1206, 1180, 1158, 1115, 1087, 1085, 1064, 1037, 1027, 971, 960, 951, 926, 902, 886, 870, 831, 820, 806, 780, 760, 740, 728, 701, 685, 668, 637, 608, 594, 567, 558, and 516 cm”1 (values rounded to the nearest whole number). This unique set of IR absorption bands or a subset thereof can be used to identify Form A.

One such subset comprises absorption bands at about 3141, 3098, 3068, 3049, 2953, 2934, 2854, 2266, 2225, 1699, 1657, 1609, 1586, 1553, 1517, 1492, 1478, 1450, 1380, 1351, 1327, 1236, 1206, 1115, 1063, 902, 886, 870, 820, 780, 760, 685, 608, 594, and 516 cm 1. Another subset comprises absorption bands at about 3141, 2953, 2934, 2854, 2266, 2225, 1699, 1657, 1450, 1206, 886, 760, 685, 594, and 516 cm 1. Yet another subset comprises absorption bands at about 3141, 2953, 2934, 2266, 1699, 1657, 1450, and 1206 cm 1.

Aprepitant case study FTIR.. READING MATERIAL http://alpha.chem.umb.edu/chemistry/ch361/spring%2005/ftir%20polymorph.pdf

Figure 5 (A-D) shows a characteristic Raman spectrum of Form A. The major Raman bands expressed in reciprocal wavelengths (wavenumber in cm”1) are positioned at about 3100, 3068, 3049, 2977, 2954, 2935, 2875, 2855, 2787, 2263, 2225, 2174, 1698, 1659, 1626, 1607, 1586, 1492, 1478, 1451, 1439, 1409, 1400, 1382, 1351, 1328, 1290, 1281, 1271, 1237, 1223, 1213, 1180, 1155, 1134, 1115, 1084, 1063, 1035, 971, 952, 926901, 868, 805, 780, 759, 740, 727, 701, 686, 669, 609, 594, 566, 558, 516, 487, 479, 433, 418, 409, 294, 274, 241, 218, 191 and 138 cm”1 (values rounded to the nearest whole number). This unique set of Raman bands or a subset thereof may be used to identify Form A.

One such subset comprises Raman bands at about 2954, 2935, 2225, 1698, 1659, and 1607 cm”1. Another subset comprises Raman bands at about 3068, 2954, 2935, 2225, 1698, 1659, 1607, 1586, 1223, 1180, 901, 780, 759, 669, and 516 cm”1. Yet another subset comprises Raman bands at about 3100, 3068, 2225, 1698, 1659, 1607, 1586, 1351, 1237, 1223, 1180, 1155, 1134, 1115, 1063, 952, 926, 901, 868, 805, 780, 759, 740, 669, 609, and 516 cm”1.

Form A was further characterized by solution 1H NMR and solid-state 13carbon NMR. The spectra are reported in Figures 6 and 7, respectively. Chemical assignments were not performed; however, the spectra are consistent with the known chemical structure of Compound I. US8084605

Example 11. Characterization of Form A Material prepared by the procedure of Example 1 was designated as Form A. The material was characterized by XRPD, TGA, DSC, hot stage microscopy, FT-IR, FT- Raman, ¹H NMR, and ¹³C NMR. The analyses were conducted according to the procedures outlined in Section B of Example 3.

The characteristic spectra and thermograms for Form A are reported in Figures 1-7. The characterization data are summarized in Table D. Table D. Characterization Data of Form A of Compound I US8084605

Amorphous solid dispersion screening

Using the amorphous form of a drug substance offers several advantages with respect to dissolution rate and solubility of the substance. However, reduced chemical stability, increased hygroscopicity and, most important, physical instability are the major drawbacks of using the amorphous phase in the final drug product. These drawbacks can be overcome by stabilizing the amorphous phase of the API in a polymer matrix, e.q. an amorphous solid dispersion. Amorphous phases dissolve more rapidly than crystalline forms, and can significantly increase bioavailability of poorly water soluble drugs substances. However, the use of amorphous materials requires confidence that crystallization will not occur during the product lifespan. For a material that has never been obtained in a crystalline form, focus should be put on attempting to crystallize it. Crystallics has extensive experience of obtaining crystalline phases from amorphous materials.

Dispersions of a drug substance onto a polymeric matrix has received increased attention in recent years. A successful dispersion results in an amorphous solid material and will show improved dissolution rates and higher apparent solubility characteristics, as well as, sufficient resistance to chemical degradation and should be physically stable e.q. sufficient high glass transition temperature avoiding crystallization of the API.

A variety of factors contribute to the formation of a suitable Amorphous Solid Dispersion (ASD), including the nature of the polymer, the drug polymer ratio, the impact of surfactants and the solvent used in the process. Crystallics has developed high-throughput solid dispersion screening technology in order to find the optimal combination of these factors.

Example 10. Preparation of Amorphous Form US8084605

A sample of Compound I (40 mg) was dissolved in 1000 μl of water. The solution was filtered through a 0.2 μm nylon filter into a clean vial then frozen in a dry ice/acetone bath. The vials were covered with a Kimwipe then placed on a lyophilizer overnight. The resulting solids yielded Amorphous Form. 8. Amorphous Form The Amorphous Form of Compound I was prepared by lyophilization of an aqueous solution of Compound I (Example 10). The residue material was characterized by XRPD and the resulting XRPD spectrum displayed in Figure 26. The XRPD spectrum shows a broad halo with no specific peaks present, which confirms that the material is amorphous. The material was further characterized by TGA, DSC, hot stage microscopy, and moisture sorption analysis.

TGA analysis (Figure 27) showed a 1.8% weight loss from 25 ⁰C to 95 ⁰C, which was likely due to loss of residual solvent.

DSC analysis (Figure 28) showed a slightly concave baseline up to an exotherm at 130 ⁰C (recrystallization), followed by an endotherm at 194 ⁰C, which results from the melting of Form A. Hot stage microscopy confirmed these recrystallization and melting events (micrographs not included). An approximate glass transition was observed (Figure29) at an onset temperature of 82 ⁰

C. 

Moisture sorption/desorption data (Figure 30 and Example 25) showed a 1.0% weight loss on equilibration at 5% relative humidity. Approximately 8% of weight was gained up to 65% relative humidity. Approximately 7% of weight was lost at 75% relative humidity. This is likely due to the recrystallization of the amorphous material to a crystalline solid. A 4.4% weight gain was observed on sorption from 75% to 95% relative humidity. Approximately 4.7% weight was lost on desorption from 95% to 5% relative humidity.

The solid material remaining after the moisture sorption analysis was determined to be Form A by XRPD (Figure 31). Table H. Characterization Data of Amorphous Form US8084605

T=temperature, RH=relative humidity, MB = moisture sorption/desorption analysis Example 19: Relative Humidity Stressing Experiments

 Moisture Sorption/Desorption Study of Amorphous Form.

Mositure sorption and desorption study was conducted on a sample of Amorphous Form. The sample was prepared by lyophilolization of a solution of Compound I in water (Example 3, section A.9). The mositure sorption and desorption study was conducted according to the procedures outlined in Example 3, section B.10. The data collected is plotted in Figure 29 and summarized in Table N .Table N. Moisture Sorption/Desorption of Amorphous Form

Table B. Crystallization Experiments of Compound I from Solvents

a) FE = fast evaporation; SE = slow evaporation; RT = room temperature; SC = slow cool; CC = crash cool, MB = moisture sorption/desorption analysis b) qty = quantity; PO = preferred orientation Table C. Crystallization Experiments of Compound I in Various Solvent/Antisolvent

a precipitated by evaporation of solvent Table A. Approximate Solubilities of Compound I US8084605

a) Approximate solubilities are calculated based on the total solvent used to give a solution; actual solubilities may be greater because of the volume of the solvent portions utilized or a slow rate of dissolution. Solubilities are reported to the nearest mg/mL.

Example 3.

Polymorph Screen Compound I as prepared by the method described in Example 1 was used as the starting material for the polymorph screen. Solvents and other reagents were of ACS or HPLC grade and were used as received. A. Sample Generation. Solids for form identification were prepared via the following methods from Compound I.

1. Fast Evaporation (FE) A solution of Compound I was prepared in test solvents. The sample was placed in the hood, uncovered, to evaporate under ambient conditions. The solids were analyzed by XRPD for form identification.

2. Slow Evaporation (SE) A solution of Compound I was prepared in test solvents. The sample was placed in the hood, covered with foil rendered with pinholes, to evaporate under ambient conditions. The solids were analyzed by XRPD for form identification.

3. Room Temperature (RT) Slurries An excess amount of Compound I was slurried in test solvent on a rotating wheel for approximately 5 or 7 days. The solids were typically collected by vacuum filtration, air dried in the hood, and analyzed by XRPD for form identification.

4. Elevated Temperature Slurries Excess Compound I was slurried in test solvents at 47 ⁰C on a shaker block for approximately 5 days. The solids were collected by vacuum filtering, dried in the hood, and then analyzed by XRPD for form identification.

5. Slow Cooling Crystallization (SC)

A saturated or near saturated solution of Compound I was prepared at elevated temperature. The samples were filtered through warmed 0.2 μm filters into warmed vials. The heat source was turned off and the samples slowly cooled to ambient temperature. If precipitation did not occur within a day the samples were placed in the refrigerator. The samples were transferred to a freezer if precipitation did not occur within several days. The solids were collected by decanting the solvent or vacuum filtration, dried in the hood and analyzed by XRPD for form identification.

6. Crash Cooling Crystallization (CC) A saturated or near saturated solution of Compound I was prepared at elevated temperature. The samples were filtered through warmed 0.2 μm filters into warmed vials then rapidly cooled in an acetone/dry ice or ice bath. If precipitation did not occur after several minutes the samples were placed in the refrigerator or freezer. Solids were collected by decanting solvent or vacuum filtration, dried in the hood, and then analyzed by XRPD. Samples that did not precipitate under subambient conditions after several days were evaporated in the hood and analyzed by XRPD for form identification.

7. Solvent/Antisolvent Crystallization (S/AS) A solution of Compound I was prepared in test solvent. A miscible antisolvent was added with a disposable pipette. Precipitate was collected by vacuum filtration or decanting solvent. The samples were stored under subambient conditions if precipitation did not occur. If solids were not observed after several days the samples were evaporated in the hood. Collected solids were analyzed by XRPD for form identification.

8. Relative Humidity (RH) Stressing Experiments Samples of Compound I were placed uncovered in approximately 58%, 88%, and 97% relative humidity jars. The samples were stored in the jars for approximately 8 days. The solids were collected and analyzed by XRPD for form identification.

9. Lyophilization Compound I was dissolved in water in a glass vial. The solution was frozen by swirling the vial in an acetone/dry ice bath. The frozen sample was placed on the lyophilizer until all of the frozen solvent was removed. The solids were collected and analyzed by XRPD for form identification.

10. Grinding Experiments Aliquots of Compound I were ground manually with a mortar and pestle as a dry solid and a wet paste in water. The samples were ground for approximately three minutes. The solids were collected and analyzed by XRPD for form identification.

11. Dehydration Experiments Hydrated samples of Compound I were dehydrated at ambient conditions (2 days) and in an ambient temperature vacuum oven (1 day). The solids were collected and analyzed by XRPD for form identification.

12. Vapor Stress Experiments Amorphous Compound I was placed in acetone, ethanol, and water vapor chambers for up to eight days. The solids were collected and analyzed by XRPD for form identification.

STABILITY STUDY Stability studies are commonly performed for new drug entities with chemical stability and impurity formation being investigated. It is also important to monitor the physical stability under these same conditions to anticipate any form changes that may occur. As an example, many hydrates will dehydrate to a lower hydrate or anhydrous form at elevated temperatures. Anhydrous materials can also undergo form transformations to other anhydrous forms upon heating.

These types of changes can be monitored using heating studies in an oven with subsequent XRPD analysis or in-situ variable temperature XRPD can be used to look for changes. In other cases, anhydrates will convert to hydrates or the API in an amorphous solid dispersion may crystallize under elevated relative humidity (RH) conditions.

Equilibration in RH chambers with subsequent analysis by XRPD or in-situ variable RH XRPD experiments can be used to readily identify these form changes. Once the effect of temperature and RH on form changes is understood, this can be factored into other processes such as drying, formulation, storage, and packaging B. Sample Characterization. The following analytical techniques and combination thereof were used determine the physical properties of the solid phases prepared.

1. X-Ray Powder Diffraction (XRPD)

XRPD is commonly used as the initial method of analysis for form screens. For polymorph, salt, and co-crystal screens XRPD is used to determine if a new form has been produced by comparing the powder pattern to all known forms of the API and the counterion/guest. If a new form is found by XRPD, additional characterization by other methods is in order. For amorphous solid dispersion screens, XRPD is used to confirm a lack of crystallinity indicated by an amorphous halo in the powder pattern.

The halos will move depending on the concentration and interactions of the API and polymer. Computational methods have also been used with XRPD data to establish miscibility of amorphous solid dispersions X-ray powder diffraction is a front line technique in solid form screening and selection based on its ability to give a fingerprint of the solid-state structure of a pharmaceutical material. Understanding the solid forms of a pharmaceutical compound provides a road map to help direct a variety of development activities ranging from crystallization, formulation, packaging, storage, and performance.

Different screening and selection strategies are warranted in early and late development because different information is needed at the various stages. Solid form selection and formulation approaches need to be investigated together and tailored to the situation. It is important to include solid form selection and possible changes in form as part of the risk management strategy throughout the drug development process.

X-ray powder diffraction (XRPD) analyses were performed using an Inel XRG- 3000 diffractometer equipped with a CPS (Curved Position Sensitive) detector with a 2Θ (2Θ) range of 120°. Real time data were collected using Cu-Ka radiation starting at approximately 4 °2Θ at a resolution of 0.03 °2Θ. The tube voltage and amperage were set to 40 kV and 30 mA, respectively. The pattern is displayed from 2.5 to 40 °2Θ. Samples were prepared for analysis by packing them into thin- walled glass capillaries. Each capillary was mounted onto a goniometer head that is motorized to permit spinning of the capillary during data acquisition. The samples were analyzed for approximately 5 minutes.

Instrument calibration was performed using a silicon reference standard. Peak picking was performed using the automatic peak picking in the Shimadzu XRD-6000 Basic Process version 2.6. The files were converted to Shimadzu format before performing the peak picking analysis. Default parameters were used to select the peaks.

2. Thermogravimetric Analysis (TGA)

Thermogravimetric (TG) analyses were performed using a TA Instruments 2950 thermogravimetric analyzer. Each sample was placed in an aluminum sample pan and inserted into the TG furnace. The furnace was first equilibrated at 25 ⁰C, then heated under nitrogen at a rate of 10 °C/min, up to a final temperature of 350 ⁰C. Nickel and Alumel™ were used as the calibration standards.

3. Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry (DSC) was performed using a TA Instruments differential scanning calorimeter 2920. The sample was placed into an aluminum DSC pan, and the weight accurately recorded. The pan was covered with a lid and then crimped. The sample cell was equilibrated at 25 ⁰C and heated under a nitrogen purge at a rate of 10 °C/min, up to a final temperature of 350 ⁰C. Indium metal was used as the calibration standard. Reported temperatures are at the transition maxima. For studies of the glass transition temperature (T_g) of the amorphous material, the sample cell was equilibrated at ambient temperature, then heated under nitrogen at a rate of 20 °C/min, up to 100 ⁰C. The sample cell was then allowed to cool and equilibrate at -20 ⁰C. It was again heated at a rate of 20 °C/min up to 100 ⁰C and then cooled and equilibrated at -20 ⁰C. The sample cell was then heated at 20 °C/min up to a final temperature of 350 ⁰C. The T_g is reported from the onset point of the transition.

4. Hot Stage Microscopy.

Hot stage microscopy was performed using a Linkam hot stage (model FTIR 600) mounted on a Leica DM LP microscope. The samples were prepared between two cover glasses and observed using a 20χ objective with crossed polarizers and first order compensator. Each sample was visually observed as the stage was heated. Images were captured using a SPOT Insight™ color digital camera with SPOT Software v. 3.5.8. The hot stage was calibrated using USP melting point standards.

5. Thermogravimetric-Infrared (TG-IR)

Thermogravimetric infrared (TG-IR) analyses were acquired on a TA Instruments thermogravimetric (TG) analyzer model 2050 interfaced to a Magna 560^® Fourier transform infrared (FT-IR) spectrophotometer (Thermo Nicolet) equipped with an Ever-Glo mid/far IR source, a potassium bromide (KBr) beamsplitter, and a deuterated triglycine sulfate (DTGS) detector. The TG instrument was operated under a flow of helium at 90 and 10 cc/min for the purge and balance, respectively. Each sample was placed in a platinum sample pan, inserted into the TG furnace, accurately weighed by the instrument, and the furnace was heated from ambient temperature to 250 ⁰C at a rate of 20 °C/min.

The TG instrument was started first, immediately followed by the FT-IR instrument. Each IR spectrum represents 32 co-added scans collected at a spectral resolution of 4 cm^“1. A background scan was collected before the beginning of the experiment. Wavelength calibration was performed using polystyrene. The TG calibration standards were nickel and Alumel™. Volatiles were identified from a search of the High Resolution Nicolet TGA Vapor Phase spectral library.

6. Fourier Transform Infrared Spectroscopy (FT-IR)

Infrared spectra were acquired on a Magna-IR 560^® or 860^® Fourier transform infrared (FT-IR) spectrophotometer (Thermo Nicolet) equipped with an Ever-Glo mid/far IR source, an extended range potassium bromide (KBr) beamsplitter, and a deuterated triglycine sulfate (DTGS) detector. A diffuse reflectance accessory (the Collector™, Thermo Spectra-Tech) was used for sampling. Each spectrum represents 256 co-added scans collected at a spectral resolution of 4 cm^“1. Sample preparation consisted of physically mixing the sample with KBr and placing the sample into a 13 -mm diameter cup. A background data set was acquired on a sample of KBr. A Log 1/R (R = reflectance) spectrum was acquired by taking a ratio of these two data sets against each other. Wavelength calibration was performed using polystyrene. Automatic peak picking was performed using Omnic version 7.2.

7. Fourier Transform Raman Spectroscopy (FT-Raman)

FT-Raman spectra were acquired on a Raman accessory module interfaced to a Magna 860^® Fourier transform infrared (FT-IR) spectrophotometer (Thermo Nicolet). This module uses an excitation wavelength of 1064 nm and an indium gallium arsenide (InGaAs) detector. Approximately 0.5 W of Nd)YVO₄ laser power was used to irradiate the sample. The samples were prepared for analysis by placing the material in a glass tube and positioning the tube in a gold-coated tube holder in the accessory. A total of 256 sample scans were collected from at a spectral resolution of 4 cm^“1, using Happ-Genzel apodization. Wavelength calibration was performed using sulfur and cyclohexane. Automatic peak picking was performed using Omnic version 7.2.

8. Solid State Nuclear Magnetic Resonance Spectroscopy (¹³C-NMR)

The solid-state ¹³C cross polarization magic angle spinning (CP/MAS) NMR spectrum was acquired at ambient temperature on a Varian ^UN1TYINOVA-400 spectrometer (Larmor frequencies: ¹³C = 100.542 MHz, ¹H = 399.799 MHz). The sample was packed into a 4 mm PENCIL type zirconia rotor and rotated at 12 kHz at the magic angle. The spectrum was acquired with phase modulated (SPINAL-64) high power ¹H decoupling during the acquisition time using a ¹H pulse width of 2.2 μs (90°), a ramped amplitude cross polarization contact time of 5 ms, a 30 ms acquisition time, a 10 second delay between scans, a spectral width of 45 kHz with 2700 data points, and 100 co-added scans. The free induction decay (FID) was processed using Varian VNMR 6.1C software with 32768 points and an exponential line broadening factor of 10 Hz to improve the signal-to- noise ratio. The first three data points of the FID were back predicted using the VNMR linear prediction algorithm to produce a flat baseline. The chemical shifts of the spectral peaks were externally referenced to the carbonyl carbon resonance of glycine at 176.5 ppm. 9. Solution Nuclear Magnetic Resonance Spectroscopy (¹H-NMR) The solution ¹H NMR spectrum was acquired at ambient temperature with a

spectrometer at a ¹H Larmor frequency of 399.803 MHz. The sample was dissolved in methanol. The spectrum was acquired with a ¹H pulse width of 8.4 μs, a 2.50 second acquisition time, a 5 second delay between scans, a spectral width of 6400 Hz with 32000 data points, and 40 co-added scans. The free induction decay (FID) was processed using Varian VNMR 6.1C software with 65536 points and an exponential line broadening factor of 0.2 Hz to improve the signal-to-noise ratio. The spectrum was referenced to internal tetramethylsilane (TMS) at 0.0 ppm. 10.

Moisture Sorption/Desorption Analysis Moisture sorption/desorption data were collected on a VTI SGA-100 Vapor Sorption Analyzer. Sorption and desorption data were collected over a range of 5% to 95% relative humidity (RH) at 10% RH intervals under a nitrogen purge. Samples were not dried prior to analysis. Equilibrium criteria used for analysis were less than 0.0100% weight change in 5 minutes, with a maximum equilibration time of 3 hours if the weight criterion was not met. Data were not corrected for the initial moisture content of the samples. NaCl and PVP were used as calibration standards

Does solid form matter? 

Sometimes the properties of two solid forms of a drug are quite similar. In other cases, the physical and chemical properties can vary dramatically and have great impact on pharmacokinetics, ease of manufacturing, and dosage form stability. Properties that can differ among solid forms of a substance include color, solubility, crystal shape, water sorption and desorption properties, particle size, hardness, drying characteristics, flow and filterability, compressibility, and density.

Different solid forms can have different melting points, spectral properties, and thermodynamic stability. In a drug substance, these variations in properties can lead to differences in dissolution rate, oral absorption, bioavailability, levels of gastric irritation, toxicology results, and clinical trial results. Ultimately both safety and efficacy are impacted by properties that vary among different solid forms. Stability presents a special concern, since it’s easy to inadvertently generate the wrong form at any point in the development process.

Because energy differences between forms are usually relatively small, form interconversion is common and can occur during routine API manufacturing operations and during drug product formulation, storage, and use. The stakes are high. Encountering a new solid form during late stages of development can delay filing. A new form appearing in drug product can cause product withdrawal.  

When should a search for solid forms begin?

The key to speed in the drug development process is to do it right the first time. For solid pharmaceuticals, that means:

• identify the optimum solid form early in drug development
• make the same form for clinical material and commercial API
• develop a crystallization process that assures control of solid form
• produce a drug product with solid form stability through expiration

scientists strongly recommend that investigation of possible solid forms of a new chemical entity be carried out as early in the development process as drug supply will allow. The best approach has three stages. The first stage, more relevant to some development processes than to others, is a milligram-scale abbreviated screen on efficacious compounds prior to final IND candidate selection. This early information can be used to guide selection of salts and solid forms for scale-up and toxicology studies. The second stage is full polymorph screening and selection of optimum solid form. This stage is important to all development processes and should certainly occur before the first GMP material is produced. In the case of ionic drugs, various salts should be prepared and screened for polymorphs and hydrates. The third stage, an exhaustive screen carried out before drug launch, is an effort to find and patent all of the forms of a high-potential drug. Staging the screening in this way optimizes the balance among the factors of early knowledge of options, probability of commercial success, and judicious investment of R&D money.

Delay in understanding solid form issues results in problems like different batches of clinical material having different solid forms. Another common and preventable dilemma arises when clinical trials are carried out with one form while commercial production generates another. In this case, bridging studies are required to demonstrate to regulatory agencies that the clinicals are relevant. ICH guidelines require a search for solid forms, comparison of properties that might affect product efficacy, and, if appropriate, setting of solid form specifications.

How is solid form controlled in API manufacture? 

It is important to control solid form during API synthesis in order to demonstrate complete process control to regulatory agencies. Different solid forms can have different solubilities and can affect recovery of API. Purification efficiencies can vary due to differential exclusion of impurities. Filtration and transfer characteristics often differ between forms. Ease of drying can vary due to different abilities to bind solvent and water in the crystal lattice. A prevalent but incorrect belief is that solid form is determined primarily by choice of crystallization solvent. In fact, it is well established that parameters like temperature, supersaturation level, rate of concentration or cooling, seeding, and ripening can have an overriding effect. These variables must be controlled to ensure consistency of solid form in API.

Can solid form problems arise in drug products, too? 

The potential for solid form variation does not end at API production. Solid form issues remain through formulation, manufacture, storage, and use of drug product. It is common to observe form transformation during standard manufacturing operations like wet granulation and milling. Excipient interactions and compaction can induce form changes. Changes can occur in the final dosage form over time. Suspensions, including those in transdermal patches, are particularly vulnerable because they provide a low-energy pathway (dissolution/recrystallization) for form interconversion. Lyophile cakes are normally amorphous, but can crystallize on storage leading to difficulty in reconstitution. Even products containing drug in solution, such as filled gel caps, can be affected if the solution is or becomes supersaturated with respect to one of the possible solid forms of the drug.

How can you tell when you have a solid form problem? 

Whenever there is a specification failure in drug product or drug substance, solid form changes should be considered in the search for causes. Particularly symptomatic is failure to meet melting point or dissolution specifications. Changes in humidity, crystallization conditions, or crystallization solvent can produce unwanted forms. Solvents known to readily produce solvates include water, alcohols, chlorinated hydrocarbons, cyclic ethers, ketones, nitriles, and amides. Changes in the appearance of gel caps or cracking of tablet coatings can indicate solid form problems. Various solid-state analytical techniques can be used to identify solid form in API. Some techniques can even determine solid form of API in intact final dosage form. Among the most useful techniques for solid-state characterization are melting point, DSC, TGA, hot stage and optical microscopy, solid-state NMR, IR and Raman spectroscopy, and X-ray powder diffraction.

Is there any good news about polymorphism? 

Polymorphism presents opportunities as well as challenges. Investigation of the properties of different forms of a commercial drug can lead to new products with improved onset time, greater bioavailability, sustained release properties, or other therapeutic enhancements. New forms can bring improvements in manufacturing costs or API purity. These improvements are patentable and can provide a competitive advantage. An underutilized potential of polymorphism is to solve formulation problems that cause the abandonment of potentially useful drugs in which much investment has already been made.

SOLUBILITY

Solubility is an important parameter for new molecules especially with the emergence of many poorly soluble compounds in the drug discovery and development pipeline. Polymorphic forms can exhibit solubility differences that vary within a factor of 1-5, amorphous solid dispersions show an improvement one or two orders of magnitude higher, and salts and co-crystals fall between these extremes . A comparison of solubility values of pure forms will provide important information when deciding on a solid form or dosage form. X-ray powder diffraction will allow identification of pure forms for these types of measurements.

However, form changes during solubility and dissolution experiments are also possible and need to be investigated. Solids remaining at the end of solubility and dissolution experiments should always be analyzed initially by XRPD to determine if a form transformation has occurred under these conditions. If a form change has occurred, XRPD patterns can be compared to known forms (polymorphs, hydrates, salts, free acid/base) in order to identify the solids remaining. If a pattern is obtained that does not correspond to known forms, complementary methods will be needed to determine properties such as hydration state or a change in stoichiometry as would be observed from breaking a salt and forming the free acid/base or the formation of salts in buffered solutions.

FORMULATION

Formulators are charged with the responsibility to formulate a product which is physically and chemically stable, manufacturable, and bioavailable. Most drugs exhibit structural polymorphism, and it is preferable to develop the most thermodynamically stable polymorph of the drug to assure reproducible bioavailability of the product over its shelf life under a variety of real-world storage conditions. There are occasional situations in which the development of a metastable crystalline or amorphous form is justified because a medical benefit is achieved. Such situations include those in which a faster dissolution rate or higher concentration are desired, in order to achieve rapid absorption and efficacy, or to achieve acceptable systemic exposure for a low-solubility drug.

Another such situation is one in which the drug remains amorphous despite extensive efforts to crystallize it. If there is no particular medical benefit, there is less justification for accepting the risks of intentional development of a metastable crystalline or amorphous form. Whether or not there is medical benefit, the risks associated with development of a metastable form must be mitigated by laboratory work which provides assurance that (a) the largest possible form change will have no substantive effect on product quality or bioavailability, and/or (b) a change will not occur under all reasonable real-world storage conditions, and/or (c) analytical methodology and sampling procedures are in place which assure that a problem will be detected before dosage forms which have compromised quality or bioavailability can reach patients.

Crystal engineering and co-crystals

Crystal engineering is generally considered to be the design and growth of crystalline molecular solids with the aim of impacting material properties. A principal tool is the hydrogen bond, which is responsible for the majority of directed intermolecular interactions in molecular solids. Co-crystals are multi-component crystals based on hydrogen bonding interactions without the transfer of hydrogen ions to form salts – this is an important feature, since Brønsted acid-base chemistry is not a requirement for the formation of a co-crystal.

Co-crystallization is a manifestation of directed self-assembly of different components. Co-crystals have been described of various organic substances over the years (33,34) and given various names, such as addition compounds (35,36) molecular complexes (37,38) and heteromolecular co-crystals (39). Regardless of naming convention, the essential meaning is that of a multi-component crystal where no covalent chemical modification of the constituents occurs as a result of the crystal formation. Pharmaceuticals co-crystals have only recently been discussed as useful materials for drug products.  

Pharmaceutical co-crystals

Pharmaceutical co-crystals can be defined as crystalline materials comprised of an active pharmaceutical ingredient (API) and one or more unique co-crystal formers, which are solids at room temperature. Co-crystals can be constructed through several types of interaction, including hydrogen bonding, p-stacking, and van der Waals forces. Solvates and hydrates of the API are not considered to be co-crystals by this definition. However, co-crystals may include one or more solvent/water molecules in the crystal lattice. An example of putative design, a construction and preparation process is shown in Figure 2 for the 5-fluororuracil:urea 1:1 co-crystal(40).

This real example neatly illustrates the opportunity and challenge that exists currently with designing pharmaceutical co-crystals. Firstly, the ‘design’ is challenging because we have no ability to predict the exact crystal structure that may result from a crystallization attempt. By analogy to the challenge of deriving protein structure from first principles, the primary sequence (chemical structure in our case) is known and elements of secondary structure (the 2-D tape construction in Figure 2) are somewhat discernible from primary information. Prediction of the actual 3-D folded conformation (tertiary structure or obtained by self-assembly) is not possible. In other words, while we currently have the ability to project which things associate in what approximate manner on the secondary level, crystal structure prediction is essentially an intractable proposition.

By extension, and just as the exact function of a protein and quantitative parameters of activity are not predictable from primary and secondary structure, the prediction of crystal properties is not possible in the absence of structural information and measurements. There is early evidence that practitioners were aware that apparent co-crystallization of drugs could lead to useful preparations (41). In fact, a ‘chemical compound’ composed of sulfathiazole and proflavin dubbed flavazole was used to treat bacterial infection during the Second World War (42).

The case of flavazole reveals insight into how two different molecules might interact in a putative co-crystal:“… flavazole is definitely a chemical compound containing equimolar proportions of sulphathiazole and proflavin base. It is believed that combination occurs through the acidic sulphonamide group (SO2NH) of the sulphathiazole and the basic centres of the proflavin. Perhaps the most realistic expression of the formula would be to place proflavin and sulphathiazole side by side with a comma between them.” (42)  In the second half of the 20th century, interest in co-crystals evolved into the directed study of intermolecular interactions in crystalline solids (43-45). The technical development of routine single-crystal structure determination led to a watershed of data, now largely accessible through the Cambridge Structural Database (CSD) (46,47).

The structural data have become useful for understanding the intermolecular interactions in co-crystals in atomic level detail (48). Using insight gained from analysis of the CSD and directed experimentation, scientists attempt design of co-crystals with specific properties, such as color or non-linear optical response, by selecting starting components with appropriate molecular properties likely to exhibit specific intermolecular interactions in a crystal (49-52).

However, even when chemically compatible functional groups are present it is not possible to accurately predict if a co-crystal, a eutectic mixture or simply a physical mixture will result from any given experiment. As a result of these complexities, attention has been directed at the identification and characterization of intermolecular packing motifs with the goal of developing principles for co-crystal materials (53).

Figure 2.  Steps involved in crystal engineering of a pharmaceutical phase, exemplified by the real example of co-crystallization of 5-fluorouracil and urea. Scientists in India have reported a rare example of synthon polymorphism in co-crystals of 4,4′-bipyridine and 4-hydroxybenzoic acid.

Polymorphism is defined as the ability of a material to exist in more than one form or crystal structure. It has important implications for the properties of such materials; for example in pharmaceuticals, the dissolution rate of a drug can be dependent on the polymorphic form. While this is a common phenomenon in single crystals it is much less common in co-crystals, systems where the structure has at least two distinct components. Gautam Desiraju from the Indian Institute of Science, found that when 4,4′-bipyridine and 4-hydroxybenzoic acid were dissolved together in a solvent such as methanol they would co-crystallise to form two different polymorphs. They noticed that a third form, a pseudopolymorph, was also present.

PROSPECTS FOR CRYSTAL ENGINEERING AND PHARMACEUTICAL CO-CRYSTALS

At the beginning of the 21st century, the field of crystal engineering has experienced significant development. Importantly, crystal engineering principles are now being actively considered for application to pharmaceuticals to modulate the properties of these valuable materials (54).

Because the physical properties that influence the performance of pharmaceutical solids are reasonably well appreciated, there is a unique opportunity to apply crystal engineering techniques and the appropriate follow-up studies to solve real world problems, such as poor physical and chemical stability or inadequate dissolution for appropriate biopharmaceutical performance of an oral drug. As structures and series of pharmaceutical co-crystals have begun to appear, we again find that properties cannot be predicted from the structures.  Nevertheless, occasional trends have been suggested.

For example, insoluble drug compounds co-crystallized with highly water soluble complements tend to achieve kinetic solubilities in aqueous media several times greater than the pure form (55,56).

There are also more possible phases for each given active compound to consider, thus there will arguably be a greater opportunities for property enhancement. In terms of stability enhancement and solubilization, the example of the series of itraconazole co-crystals with pharmaceutically acceptable 1,4-diacids (55) suggests a strategy alternative to amorphous drug formulation. The co-crystal options presented retain the stability inherent in a crystalline state, while allowing for solubilization that significantly exceeds that of crystalline itraconazole base and rivals the performance of the engineered amorphous bead formulation (Sporanox®).

Where are we now? From recent literature it appears that knowledge gained over the past century and increasingly sophisticated screening techniques developed within the last decade are paving the way towards design of co-crystals with potentially improved pharmaceutical properties (55-58) In terms of the application to pharmaceutical systems, the field of crystal engineering is developing the retro-synthetic understanding of crystal structure using reasoning that is analogous to that applied by organic chemists.  For example, the retro-synthetic approach in covalent synthesis operates on the level of a single molecule, while the analogous effort in crystal engineering focuses on the “supermolecule”:

piracetam

The assemblies that define the crystalline arrangement of the molecules as they self-organize into the solid-state. The parallels between the development of crystal engineering and synthetic organic chemistry run still deeper. Methodologies for carrying out these crystallizations are being developed alongside the development of new robust motifs (6,53,55,57,60). The importance of the solubility and dissolution relationships of the components of a putative co-crystal is becoming a matter of significant investigation (56,60). The same can be said for the roles of additives in templating novel forms.

Mechanical milling of materials has also been documented as a means to make co-crystals, and a recent example of polymorphic forms of caffeine:glutaric acid illustrates the opportunities of this type of processing to influence crystal form (61). With an increase in the understanding of the modes of self-assembly, one can start to address the design aspect towards making pharmaceutical co-crystals.

There remain several limitations to the application of what is currently known to the design of useful materials. As mentioned earlier, it remains intractable to reliably predict crystal structure.  Multi-component crystals are well out of reach for prediction due in part to complex energetic landscapes, lack of appropriate charge density models and a large number of degrees of freedom, making computation unfeasible. Moreover, there is only a qualitative understanding of the interplay between intermolecular interactions and materials performance, especially for properties relevant to pharmaceuticals such as solubility, dissolution profile, hygroscopicity and melting point.

But the saving grace of the co-crystal approach comes in two guises: Complementarity and diversity. On the topic of complementarity, it is possible, by way of CSD database mining for instance, to identify trends of hetero-synthon occurrence in model systems. As for the diversity aspect, the space of possible co-crystal formers is large, limited only by pharmaceutical acceptability. Coupled with parameters such as stoichiometry variation and increase in the number of components (binary systems can be expanded into ternary ones, etc.), the opportunities appear vast.

THE FUTURE OF CRYSTAL ENGINEERING IN PHARMACEUTICAL SCIENCE

READ

Novel Challenges in Crystal Engineering: Polymorphs and New …

Where are we going?  At this point, we have only just scratched the surface of materials science-driven pharmaceutical product design. In the 21st century, practitioners of pharmaceutical chemistry need to enumerate and exploit the opportunities of crystal form design that nature affords us, and thus gain increasing ability to design the materials we need from the molecules that we seek to convert into pharmaceuticals.

Learning will be facilitated by advances in crystallization automation (6,62), microscopy-spectroscopy techniques (Raman and IR microscopy) and new techniques such as terahertz spectroscopy and AFM, along with increasingly sophisticated X-ray diffraction lab instrumentation. In addition, further enhancements in the data mining tools associated with the CSD operating on an ever increasing number of high-quality crystal structures will undoubtedly lead to new knowledge and principles of interaction.

The challenge placed before pharmaceutical scientists, now and in the future, is the following: (i) to understand the requirement of a particular compound in terms of materials structure and properties, and (ii) to creatively integrate crystal engineering within the limits of pharmaceutical acceptability of components to obtain new forms of active ingredients with desirable properties for formulation and delivery. It should become the collective mantra of medicinal chemists, process engineers and pharmaceutical scientists to “design and make the material we need.” This mantra can form the common aspiration for an industry that is in significant need of innovation and productivity enhancement.

Applications and Advantages

Applications

• Drug companies can use this technology to protect themselves against others generating and patenting polymorphs

Advantages

• Having more than one solid form of a drug allows optimization of drug dissolution behavior and shelf life

IP AND POLYMORPHS

In order for a new drug to enter the market, pharmaceutical companies must invest for many years in very expensive clinical trials and a lengthy regulatory approval process.Market protection plays therefore a major role in the growth of the pharmaceutical industry and Intellectual Property (IP) laws are intended to give the investors an opportunity to recover their costs. Patent filing is one way of efficiently protecting various aspects of an innovative drug.

The duration of the new drug’s market protection is, however, limited in time and once the original drug is no longer protected, legal copies (generic medicines) can be developed and marketed by competitors at more accessible prices, since the expensive basic research, as well as pre-clinical and clinical trials (at least for small molecules) are no longer necessary. Generic medicines are either identical copies of the original drug or so-called bioequivalent versions of it.

Bioequivalent means that they behave as the original drug when administered to patients. For a generic drug to be a bioequivalent its Active Pharmaceutical Ingredient (API) does not need to be the same solid form as in the original drug. A different polymorph or a pseudo-polymorph (i.e. solvate, hydrate) of the API and different excipients are acceptable variants, as long as the final generic drug product behaves as the original one.  

Solid Forms Screening

Screening of solid forms, in particular polymorphs of APIs, is therefore an essential part of pharmaceutical development and lifecycle management, not only for scientific and regulatory reasons, but also because of the key role that pharmaceutical solid forms play in the area of IP, for innovators as well as for generic companies. The knowledge generated by conducting solid form screening, in fact, can provide an innovator company the opportunity to build a strong patent portfolio around different solid forms and therefore a way to maximize returns from drug development.

This allows innovator companies to gain several years of additional protection for their product after the expiry of the basic molecule patent, since various pharmaceutical solid forms are individually patentable. In the US, for instance, innovator companies are required to identify, in the so-calledOrange Book, their patents covering different solid forms performing the same as the product described in the corresponding NDA. In return, they can benefit from a 30-months stay over a generic company which would eventually file an ANDA with aParagraph IV Certification for any of these listed patents.

 

Thus, by patenting a maximum number of possible solid forms, even if these are not further developed and used, innovator companies can more efficiently protect their own products. Such patents must obviously meet the same patentability criteria as other inventions. Conversely, a generic company can launch its own product if, after the basic molecule patent has expired, it discovers a new solid form, i.e. a form with no IP protection and suitable characteristics for product development.

In both cases, a very sensitive tool as SR-XRPD can play a key role in helping the detection and characterization of a maximum number of polymorphs.

 Accurate and direct characterization of the API polymorphic forms and detection of trace amounts has proven to be of paramount importance (e.g. Paxil^®, Cefdinir) whereas poorly conducted screens and unsuccessful patenting strategies, on the other hand, can have significant negative commercial consequences (e.g. Ritonavir).

Interestingly, in the US the first ANDA approved by FDA with paragraph IV certification is entitled to 180-days marketing exclusivity. Initially granted only when theANDA applicant having filed Paragraph IV Certification could prevail in the litigation with the originator, the new FDA guidance suppresses the “successul defence”requirement and the 180-days exclusivity is decided on a case-by-case basis and can therefore be granted even if the case is settled.

While there has been much discussion by policymakers and stakeholders about the effects of “secondary patents” on the pharmaceutical industry, there is no empirical evidence on their prevalence or determinants. Characterizing the landscape of secondary patents is important in light of recent court decisions in the U.S. that may make them more difficult to obtain, and for developing countries considering restrictions on secondary patents.

It is seen the claims of the 1304 Orange Book listed patents on all new molecular entities approved in the U.S. between 1988 and 2005, and coded the patents as including chemical compound claims (claims covering the active molecule itself) and/or one of several types of secondary claims. It is seen that  distinguish between patents with any secondary claims, and those with only secondary claims and no chemical compound claims (“independent” secondary patents).

It is seen  that secondary claims are common in the pharmaceutical industry. It is seen that independent secondary patents tend to be filed and issued later than chemical compound patents, and are also more likely to be filed after the drug is approved. When present, independent formulation patents add an average of 6.5 years of patent life (95% C.I.: 5.9 to 7.3 years), independent method of use patents add 7.4 years (95% C.I.: 6.4 to 8.4 years), and independent patents on polymorphs, isomers, prodrug, ester, and/or salt claims add 6.3 years (95% C.I.: 5.3 to 7.3 years). evidence that late-filed independent secondary patents are more common for higher sales drugs

Polymorph quantification. REF 79

SEE A SLIDESHARE PRESENTATION

http://www.slideshare.net/reychemist/polymorph-quant

The ability to detect and quantify polymorphism of pharmaceuticals is critically important in ensuring that the formulated product delivers the desired therapeutic properties because different polymorphic forms of a drug exhibit different solubilities, stabilities and bioavailabilities. The purpose of this study is to develop an effective method for quantitative analysis of a small amount of one polymorph within a binary polymorphic mixture. Sulfamerazine (SMZ), an antibacterial drug, was chosen as the model compound. The effectiveness and accuracy of powder X-ray diffraction (PXRD), Raman microscopy and differential scanning calorimetry (DSC) for the quantification of SMZ polymorphs were studied and compared.

Low heating rate in DSC allowed complete transformation from Form I to Form II to take place, resulting in a highly linear calibration curve. Our results showed that DSC and PXRD are capable in providing accurate measurement of polymorphic content in the SMZ binary mixtures while Raman is the least accurate technique for the system studied.

DSC provides a rapid and accurate method for offline quantification of SMZ polymorphs, and PXRD provides a non-destructive, non-contact analysis.A novel method of detecting very low levels of different polymorphs using high-resolution X-ray powder diffraction with a synchrotron light source has been developed by Zach-Zambon Chemicals of Italy. Key to the project has been development of software to enable appropriate data presentation.The issue of polymorphism in pharmaceuticals has attracted increasing attention over the past 20 years and is something to which development scientists and the regulatory authorities pay considerable attention.

http://www.scientificupdate.co.uk/blog/item/polymorph-detection-and-quantification-a-novel-method.html

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9. Margiolaki, I., Wright, J. P., Fitch, A. N., Fox, G. C. & Von Dreele, R. B.: Synchrotron X-ray powder diffraction study of hexagonal turkey egg-white lysozyme, Acta Cryst. D61, 423–432 (2005). See also: Margiolaki, I. & Wright, J. P.: Powder crystallography on macromolecules, Acta Cryst. A64, 169–180 (2008).
10. Fundamentals of Crystallography,C. Giacovazzo Ed., International Union of Crystallography, Oxford Science Publications, Third Edition (2011).
11. The basics of Crystallography and Diffraction (Third edition, 2010), Christopher Hammond, IUCr, Oxford Science Publications; ISBN 978-0-19-954645-9. See also: X-Ray Structure Determination, A practical Guide, George H. Stout and Lyle H. Jensen, Wiley Interscience.
12. Bruni G, Gozzo F, Capsoni D, Bini M, Macchi P, Simoncic P, Berbenni V., Milanese C., Girella A., Ferrari S. and Marini A., Thermal, Spectroscopic, and Ab Initio Structural Characterization of Carprofen Polymorphs, J. Pharm. Sciences 100(6), 2321 (2011).
13. Brunelli, M., Wright, J. P., Vaughan, G. B. M., Mora, A. J. & Fitch, A. N.: Solving Larger Molecular Crystal Structures from Powder Diffraction Data by Exploiting Anisotropic Thermal Expansion. Angew. Chem. (2003) 115, 2075–2078.
14. T. Wessels, Ch. Baerlocher and L.B. McCusker: Single-crystal-like diffraction data from polycrystalline materials, Science (1999), 284, 477-479.
15. Shankland, K., David, W. I. F., Csoka, T. & McBride, L.: Structure solution of Ibuprofen from powder diffraction data by the application of a genetic algorithm combined with prior conformational analysis,  Intl. J. Pharmaceut. (1998) 165, 117–126.
16. A. Altomare, C. Cuocci, C. Giacovazzo, A. Moliterni and R. Rizzi: The dual-space resolution bias correction algorithm: applications to powder data, J. Appl. Cryst. (2010). 43, 798-804.
17. G.Oszlanyi and A. Suto: The charge flipping algorithm, Acta Cryst. (2008). A64, 123–134 and references herein.
18. Boccaleri, E., Carniato, F., Croce, G., Viterbo, D., van Beek, W., Emerich H. and Milanesio, M.,In-situ simultaneous Raman/high-resolution X-ray powder diffraction study of transformations occurring in materials at non-ambient conditions, J. Appl. Cryst., 2007, 40, 684-693.
19. Scarlett N.V.Y. and Madsen I. C., Quantification of phases with partial or no known crystal structures, Powder Diffraction (2006) 21, 278-284
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21. Scardi, P.; Leoni, M.: Line profile analysis: pattern modeling versus profile fitting, J. Appl. Cryst. 39 (2006) 24–31. Scardi, P.; Leoni, M.: Whole Powder Pattern Modelling, Acta Crystall. A58 (2002) 190–200
22. Local structure from total scattering and atomic pair distribution function (PDF) analysis, In Powder diffraction: theory and practice, (Royal Society of Chemistry, London England, 2008), Robert E. Dinnebier and Simon J. L. Billinge, Eds., pp. 464 – 493.
23. Neder R. B. And Korsunskiy V. I., Structure of nanoparticles from powder diffraction data using the pair distribution function, 2005 J. Phys.: Condens. Matter 17 S125
24. A. Cervellino, C. Giannini, A. Guagliardi, Determination of nanoparticle, size distribution and lattice parameter from x-ray data for monoatomic materials with f.c.c. cubic unit cell, J. Appl. Cryst. 36, 1148-1158 (2003).
25. P.W. Stephens, D.E. Cox, and A.N. Fitch, Synchrotron Radiation Powder Diffraction in Structure Determination by Powder Diffraction, pp. 49-87, edited by W.I.F. David, K. Shankland, L.B. McCusker, and C. Baerlocher, (Oxford University Press, 2002)
26. Joel Bernstein: Polymorphism in Molecular Crystals, IUCr Monographs on Crystallography (2002), Oxford Science Publications.
27. Polymorphism in Pharmaceutical Solids, Ed. By Harry G. Brittain, Drugs and The Pharmaceutical Sciences, Vol. 192
28. Tremayne, M.: The impact of powder diffraction on the structural study of organic materials., Philosophical Transactions of the Royal Society of London Series A: Chemistry and Life Sciences Triennial Issue, 362: 2691 (2004).
29. Law D, Schmitt EA, Marsh KC, Everitt EA,Wang W, Fort JJ, Krill SL, Qiu Y. Ritonavir-PEG 8000 amorphous solid dispersions: in vitro and in vivo evaluations. J. Pharm. Sci. 2004; 93 (3): 563–567.
30. Bruni G., Berbenni V., Milanese C., Girella A., Cardini A., Lanfranconi S. and Marini A.,Determination of the nateglinide polymorphic purity through DSC, J. Pharm. and Biomed. Anal. 54 (2011) 1196-1199.
31. Aaltonen J., Alleso M., Mirza S., Koradia V., Gordon K.C and Rantanen J., Solid form screening – A review. European Journal of Pharmaceutics and Biopharmaceutics 71 (2009), 23-37.
32. Srivastava D., The Food and Drug Administration and Patent Law at a Crossroads: The Listing of Polymorph Patents as a Barrier to Generic Drug Entry. Food and Drug Law Journal, Vol. 59, No.2 (2004), 339-354.
33. Grabowski H. G., Kyle M., Mortimer R., Long G. & Kirson N., Evolving Brand-name And Generic Drug Competition May Warrant A Revision Of The Hatch-Waxman Act. Health Affairs, 30, no.11 (2011):2157-2166.
34. Rakowski W. A and Mazzochi D. M., The case of disappearing polymorph: ‘Inherent anticipation’ and the impact of Smithkline Beecham Corp. v Apotex Corp. (Paxil®) on patent validity and infringement by inevitable conversion’. Journal of Generic Medicine, Vol.1, No.2 (2006):131-139.
35. Cabri W., Ghetti P., Pozzi G. and Alpegiani M., Polymorphisms and Patent, Market, and Legal Battles: Cefdinir Case Study. Organic Process Research & Development 2007, 11, 64-72.
36. Bauer J, Spanton S, Henry R, Quick J, Dziki W, Porter W, Morris J., Ritonavir: an extraordinary example of conformational polymorphism, Pharm Res. 2001 Jun;18(6):859-66.

79………..related to estimation

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8. Campbell Roberts, S.N., Williams A.C., Grimsey, I.M., and Booth S.W., Quantitative analysis of mannitol polymorphs. X-ray powder diffractometry—exploring preferred orientation effects J. Pharm. Biomed. Anal., 2002, 28, 1149-1159.

US-based Biogen Idec has received approval from the European Commission (EC) for its Tecfidera (dimethyl fumarate) as a first-line oral treatment for people with relapsing-remitting multiple sclerosis (RRMS), the most common form of multiple sclerosis (MS).

Biogen’s multiple sclerosis drug Tecfidera obtains EU approval  click here

Earlier this week, Ligand Pharmaceuticals Inc. ( LGND ) announced that the U.S. Food and Drug Administration (FDA) granted breakthrough therapy designation to Promacta (EU trade name: Revolade). Ligand and its partner GlaxoSmithKline ( GSK ) are looking to get Promacta approved for the treatment of cytopenias in patients suffering from severe aplastic anemia (SAA), who are unresponsive to immunosuppressive therapy.

The FDA granted breakthrough therapy designation to Promacta based on data from an open-label phase II National Institute of Health (NIH) study (n = 43) evaluating Promacta in treatment experienced SAA patients,  who showed insufficient response to immunosuppressive therapy.

The designation, which was enacted as part of the 2012 Food and Drug Administration Safety and Innovation Act, is granted to potential new treatments for serious or life-threatening diseases or conditions where the initial clinical data shows that the treatment has the potential to demonstrate substantial improvement on one or more clinically significant endpoints compared to existing treatments. The designation should help fasten the development and review process for the candidate.

We note that Promacta is already approved for the treatment of thrombocytopenia (reduced platelet count) in patients with chronic hepatitis C virus (HCV) infection to enable the initiation and maintenance of interferon-based therapy. Promacta is also approved for thrombocytopenia in patients with chronic idiopathic thrombocytopenia (ITP).

PROMACTA (eltrombopag) Tablets contain eltrombopag olamine, a small molecule thrombopoietin (TPO) receptor agonist for oral administration. Eltrombopag interacts with the transmembrane domain of the TPO receptor (also known as cMpl) leading to increased platelet production. Each tablet contains eltrombopag olamine in the amount equivalent to 12.5 mg, 25 mg, 50 mg, 75 mg, or 100 mg of eltrombopag free acid.

Eltrombopag olamine is a biphenyl hydrazone. The chemical name for eltrombopag olamine is 3′-{ (2Z)-2-[1 -(3,4-dimethylphenyl)-3-methyl-5-oxo- 1,5-dihydro-4H-pyrazol-4- ylidene]hydrazino}-2′-hydroxy-3-biphenylcarboxylic acid – 2-aminoethanol (1:2). It has the molecular formula C₂₅H₂₂N₄O₄•2(C₂H₇NO). The molecular weight is 564.65 for eltrombopag olamine and 442.5 for eltrombopag free acid. Eltrombopag olamine has the following structural formula:

Eltrombopag olamine is practically insoluble in aqueous buffer across a pH range of 1 to 7.4, and is sparingly soluble in water.

The inactive ingredients of PROMACTA are: Tablet Core: magnesium stearate, mannitol, microcrystalline cellulose, povidone, and sodium starch glycolate. Coating: hypromellose, polyethylene glycol 400, titanium dioxide, polysorbate 80 (12.5 mg tablet), FD&C Yellow No. 6 aluminum lake (25 mg tablet), FD&C Blue No. 2 aluminum lake (50 mg tablet), Iron Oxide Red and Iron Oxide Black (75 mg tablet), or Iron Oxide Yellow and Iron Oxide Black (100 mg tablet).

Eluxadoline

5 JAN 2014

Furiex Pharmaceuticals Inc.  more than doubled in its best day of trading after its experimental drug alleviated diarrhea and abdominal pain caused by irritable bowel syndrome in two studies.

The drug eluxadoline met targets for improvements in stool consistency and abdominal pain that were developed in conjunction with U.S. and European regulators, the company said today. Furiex will apply for approval in June, Chairman Fred Eshelman said in an investor call today. He estimated annual sales of $750 million to $1 billion.

“By our math, it looks like a pretty doggone good market,” Eshelman said on the call, noting that there is only one currently approved drug available in the U.S. for the condition.

Diarrhea-predominant irritable bowel syndrome is a chronic disorder that affects about 28 million patients in the U.S. and Europe, Furiex said in the statement.Furiex said it would apply by mid-year for U.S. approval of the drug, eluxadoline, to treat diarrhea-predominant irritable bowel syndrome (IBS-d), a debilitating bowel disorder that affects about 28 million people in the United States and major European markets.

Furiex said it expected to seek European approval in early 2015.

“We believe that there are a lot of patients out there who need this drug. There is a huge unmet need,” Furiex Chief Medical Officer June Almenoff said in a telephone interview.

Currently approved drugs for IBS address constipation associated with the disorder, but there are few options for diarrhea predominant IBS.

Furiex founder and chairman Fred Eshelman said he believes the drug has the potential for blockbuster sales, which he defined as annual sales of between $750 million and $1 billion.

Eluxadoline was tested at two doses against a placebo over the course of 12 weeks to meet requirements by the U.S. Food and Drug Administration, and for 26 weeks for European health regulators, in Phase III studies involving 2,428 patients, Furiex said.

For the combined goal of improvement in abdominal pain and stool consistency for at least half the days in the study, eluxadoline achieved a statistically significant improvement at the 100 milligram and 75 mg doses through 12 weeks in both studies.

On the 26-week measure, the higher dose succeeded in both studies but the lower dose missed statistical significance in one of the two trials, according to initial results released by the company.

The success appeared to be driven by the percentage of patients reporting improvements in diarrhea, which ranged from 30 percent to 37 percent versus 22 percent and 20.9 percent for the placebo groups.

When the composite goal was broken into its two components, researchers found a numerical improvement in pain response rates that did not achieve statistical significance.

The drug appeared to be safe and well-tolerated in both studies, Furiex said. The most commonly reported side effects were constipation and nausea.

The company plans to present a far more detailed analysis of the late stage studies at an upcoming medical meeting.

“We’re very excited about the path ahead and about how this can transform patients’ lives,” Almenoff said.

Eluxadoline 

5-({[(2S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)propanoyl][(1S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl]amino}methyl)-2-methoxybenzoic acid

5-({[2-Amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid

864821-90-9 CAS

JNJ-27018966

Molecular Formula: C₃₂H₃₅N₅O₅

Molecular Weight: 569.6508

Agents for Irritable Bowel Syndrome, mu-Opioid Agonists, delta-Opioid Antagonists

Mu Delta is a locally active mu opioid receptor agonist and delta opioid receptor antagonist in phase III clinical evaluation at Furiex Pharmaceuticals for the oral treatment of diarrheal predominant irritable bowel syndrome (d-IBS).

The product candidate holds an advantage over currently marketed products for this indication because it acts locally on the enteric nervous system, possibly decreasing adverse effects on the central nervous system. In 2011, fast track designation was assigned in the U.S. for the treatment of d-IBS. In 2011, Mu Delta was licensed to Furiex Pharmaceuticals by Janssen for the treatment of d-IBS, granting an option to Furiex to continue development and commercialization following phase II proof of concept studies.

The opioid receptors were identified in the mid-1970′s, and were quickly categorized into three sub-sets of receptors (mu, delta and kappa). More recently the original three types of receptors have been further divided into sub-types. Also known is that the family of opioid receptors are members of the G-protein coupled receptor (GPCR) super-family. More physiologically pertinent are the well established facts that opioid receptors are found throughout the central and peripheral nervous system of many mammalian species, including humans, and that modulation of the respective receptors can elicit numerous, albeit different, biological effects, both desirable and undesirable (D. S. Fries, “Analgesics”, inPrinciples of Medicinal Chemistry, 4th ed.; W. O. Foye, T. L. Lemke, and D. A. Williams, Eds.; Williams and Wilkins: Baltimore, Md., 1995; pp. 247-269; J. V. Aldrich, “Analgesics”, Burger’s Medicinal Chemistry and Drug Discovery, 5^thEdition, Volume 3: Therapeutic Agents, John Wiley & Sons, Inc., 1996, pp. 321-441). In the most current literature, the likelihood of heterodimerization of the sub-classes of opioid receptors has been reported, with respective physiological responses yet undetermined (Pierre J. M. Riviere and Jean-Louis Junien, “Opioid receptors: Targets for new gastrointestinal drug development”, Drug Development 2000, pp. 203-238).

A couple biological effects identified for opioid modulators have led to many useful medicinal agents. Most significant are the many centrally acting mu opioid agonist modulators marketed as analgesic agents to attenuate pain (e.g., morphine), as well as peripherally acting mu agonists to regulate motility (e.g., loperamide). Currently, clinical studies are continuing to evaluate medicinal utility of selective delta, mu, and kappa modulators, as well as compounds possessing combined sub-type modulation. It is envisioned such explorations may lead to agents with new utilities, or agents with minimized adverse side effects relative to currently available agents (examples of side effects for morphine includes constipation, respiratory depression, and addiction potential). Some new GI areas where selective or mixed opioid modulators are currently being evaluated includes potential treatment for various diarrheic syndromes, motility disorders (post-operative ileus, constipation), and visceral pain (post operative pain, irritable bowel syndrome, and inflammatory bowel disorders) (Pierre J. M. Riviere and Jean-Louis Junien, “Opioid receptors: Targets for new gastrointestinal drug development” Drug Development, 2000, pp. 203-238).

Around the same time the opioid receptors were identified, the enkephalins were identified as a set of endogenous opioid ligands (D. S. Fries, “Analgesics”, inPrinciples of Medicinal Chemistry, 4th ed.; W. O. Foye; T. L. Lemke, and D. A. Williams, Eds.; Williams and Wilkins: Baltimore, Md., 1995; pp. 247-269). Schiller discovered that truncating the original pentapeptide enkephalins to simplified dipeptides yielded a series of compounds that maintained opioid activity (Schiller, P. WO 96/06855). However one potential drawback cited for such compounds is the likelihood of their inherent instability (P. W. Schiller et al., Int. J. Pept. Protein Res. 1993, 41 (3), pp. 313-316).

More recently, a series of opioid pseudopeptides containing heteroaromatic or heteroaliphatic nuclei were disclosed, however this series is reported showing a different functional profile than that described in the Schiller works. (L. H. Lazarus et al., Peptides 2000, 21, pp. 1663-1671).

Most recently, works around morphine related structures were reported by Wentland, et al, where carboxamido morphine derivatives and it’s analogs were prepared (M. P. Wentland et al., Biorg. Med. Chem. Letters 2001, 11, pp. 1717-1721; M. P. Wentland et al., Biorg. Med. Chem. Letters 2001, 11, pp. 623-626). Wentland found that substitution for the phenol moiety of the morphine related structures with a primary carboxamide led anywhere from equal activities up to 40 fold reduced activities, depending on the opioid receptor and the carboxamide. It was also revealed that any additional N-substitutions on the carboxamide significantly diminished the desired binding activity.

Compounds of the present invention have not been previously disclosed and are believed to provide advantages over related compounds by providing improved pharmacological profiles.

Opioid receptor modulators, agonists or antagonists are useful in the treatment and prevention of various mammalian disease states, for example pain and gastrointestinal disorders such as diarrheic syndromes, motility disorders including post-operative ileus and constipation, and visceral pain including post-operative pain, irritable bowel syndrome and inflammatory bowel disorders.

It is an object of the present invention to provide opioid receptor modulators. It is a further object of the invention to provide opioid receptor agonists and opioid receptor antagonists. It is an object of the present invention to provide opioid receptor ligands that are selective for each type of opioid receptor, mu, delta and kappa. It is a further object of the present invention to provide opioid receptor ligands that modulate two or three opioid receptor types, mu, delta and kappa, simultaneously.

It is an object of the invention to provide certain instant compounds that are also useful as intermediates in preparing new opioid receptor modulators. It is also an object of the invention to provide a method of treating or ameliorating a condition mediated by an opioid receptor. And, it is an object of the invention to provide a useful pharmaceutical composition comprising a compound of the present invention useful as an opioid receptor modulator.

5-({[2-Amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1 h-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid is an opoid receptor modulator (mu receptor agonist and delta receptor antagonist) and may be useful for treating irritable bowel syndrome, pain or other opioid receptor disorders.

5-({[2-Amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1h-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid and methods of making this molecule are disclosed in

US application 2005/02033143. Example 9 of US application 2005/02033143 makes the hydrochloride salt of 5-({[2-amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1h-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid.

Applicants have discovered a process of making the zwitterion of 5-({[2-amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1h-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid and two novel crystals of this zwitterion. In Applicant’s hands, these novel crystals provide improved properties and can be purified at higher purity. Applicant’s new process results in improved and less costly process manufacturing conditions than the procedure disclosed in US application 2005/02033143.

………………..

FIG. 6 is the molecular structure of the zwitterion 5-({[2-amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1h-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid.

US7994206

…………………..

SYNTHESIS OF 5-formyl-2- methoxy-benzoic acid methyl ester

WO2002022612A1

Example 8: 2-Methoxy-5-formylbenzoic acid

Lithium hydroxide (1.04g, 0.043mol, 3eq) in water (lOmL) was added to a stirred solution of methyl 2-methoxy-5-formylbenzoate (2.8g, 0.014mol, leq) in a mixture of tetrahydrofuran (30mL) and methanol (20mL). The solution was stirred overnight, acidified to pH 1 with 10% HCl and the organic solvents removed in vacuo. The aqueous solution was extracted with ethyl acetate (lOOmL) and the organic solution washed with brine (lOOmL), then extracted with saturated aqueous sodium bicarbonate (3 x lOOmL). The basic solution was washed with ethyl acetate (lOOmL), then acidified to pH 1 with 10% HCl and back extracted with dichloromethane (3 x lOOmL). The organic solution was dried over sodium sulfate and evaporated in vacuo to give a cream coloured powder (2.01g, 77%). 1H NMR (CDC1₃) δ 9.99 (s, IH, O=C- H), 4.14 (s, 3H, CH₃).

………………

ANALOGOUS METHOD TO PREPARE..2-methoxy-5-{[1 -(4-phenyl-1 H-imidazol-2-yl)- ethylamino]-methyl}-benzoic acid methyl ester

USE 5-formyl-2- methoxy-benzoic acid methyl ester  for 3,4- dimethoxybenzaldehyde, TO GET 2-methoxy-5-{[1 -(4-phenyl-1 H-imidazol-2-yl)- ethylamino]-methyl}-benzoic acid methyl ester 

Example 4

(3,4-Dimethoxy-benzyl)-[1-(4-phenyl-1 H-imidazol-2-yl)-ethyl]-amine

A solution of 1-(4-phenyl-1 W-imidazol-2-yl)-ethylamine (0.061 g, 0.33 mmol) of Example 3, and 0.55 g (0.33 mmol) of 3,4-dimethoxybenzaldehyde in 5 ml_ of anhydrous methanol was stirred at room temperature for 1 h and then cooled to about 0-10⁰C in an ice bath for 1 h. The reaction was treated carefully with 0.019 g (0.49 mmol) of sodium borohydride in one portion and maintained at about 0-10⁰C for 21 h. Cold 2M aqueous HCI was added dropwise (30 drops), the mixture was stirred for 5 min, and then partially concentrated in vacuo unheated. The residual material was taken up in EtOAc to yield a suspension that was treated with 5 ml_ of cold 3M aqueous NaOH and stirred vigorously until clear. The phases were separated and the aqueous layer was extracted three times additional with EtOAc. The combined extracts were dried over MgSO₄, filtered, and concentrated to yield (3,4-dimethoxy- benzyl)-[1-(4-phenyl-1 H-imidazol-2-yl)-ethyl]-amine as a light yellow oil (HPLC: 87% @ 254nm and 66% @ 214 nm).

MS (ES⁺) (relative intensity): 338.1 (100) (M+1)

This sample was of sufficient quality to use in the next reaction without further purification.

…………………..

SYNTHESIS

WO2006099060A2

In an embodiment, the present invention is directed to processes for the preparation of the compound of formula (IV)

also known as, 5-({[2-amino-3-(4-carbamoyl-2,5-dimethyl-phenyl)- propionyl]-[1 -(4-phenyl-1 H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy- benzoic acid

Example 1

(S)-2-ferf-Butoxycarbonylamino-3-(4-carbamoyl-2.6-dimethyl-phenyl)- propionic acid

STEP A: Trifluoromethanesulfonic acid 4-bromo-3,5-dimethyl-phenyl ester

To a cooled (0°C) solution of 4-bromo-3,5-dimethylphenol (3.05 g, 15.2 mmol) in pyridine (8 ml_) was added trifluoromethanesulfonic anhydride (5.0 g, 17.7 mmol) dropwise. After completion of addition, the resulting mixture was stirred at 0°C for 15 min, and then at room temperature overnight. The reaction was quenched by addition of water, and then extracted with EtOAc. The organic extracts were washed sequentially with water, 2N HCI (2x ), brine, and then dried over MgSO₄. Filtration and evaporation to dryness yielded compound 1 b as a colorless oil.

¹H NMR (300 MHz, CDCI₃): δ 2.45 (6H, s), 7.00 (2H, s).

Step B: 4-Bromo-3,5-dimethylbenzoic acid

Into a solution of compound 1 b (6.57 g, 19.7 mmol) in DMF (65 ml_) were added K₂CO₃ (13.1 g, 94.7 mmol), Pd(OAc)₂ (0.44 g, 1.97 mmol) and 1 ,1′-bis(diphenylphosphino)ferrocene (2.29 g, 4.14 mmol). The resulting mixture was bubbled in gaseous CO for 10 min and was heated to 60°C for 7.5h with a CO(₉) balloon. The cooled mixture was partitioned between aqueous NaHCO₃ and EtOAc, and filtered. The aqueous phase was separated, acidified with aqueous 6N HCI, extracted with EtOAc, and then dried over Na₂SO₄. Filtration and concentration of the filtrate yielded crude compound 1c as a brown residue, which was used in the next step without further purification. STEP C: Method A: 4-Bromo-3,5-dimethyl-benzamide

Into a suspension of compound 1c in DCM (40 ml_) was added SOCI₂ (3.1 rnL, 42 mmol) and the mixture was heated at reflux for 2 h. Upon removal of the solvent by evaporation, the residue was dissolved in DCM (40 ml_) and then ammonium hydroxide (28% NH₃ in water, 2.8 ml_) was added. The reaction mixture was heated at 5O⁰C for 2 h and concentrated. The residue was diluted with H₂O, extracted with EtOAc, and the organic portion was dried over Na₂SO₄. After filtration and evaporation, the residue was purified by flash column chramotagraphy (eluent: EtOAc) to yield compound 1 d as an off-white solid.

¹H NMR (300 MHz, CD₃CN): δ 2.45 (6H, s), 5.94 (1 H, br s), 6.71 (1 H, br s), 7.57 (2H, s)

MS(ES⁺)(relative intensity): 228.0 (100%) (M+1).

Step C: Method B: 4-Bromo-3,5-dimethyl-benzamide

A mixture of compound 1 b (3.33 g, 10 mmol), PdCI₂ (0.053 g, 0.3 mmol), hexamethyldisilazane (HMDS, 8.4 ml_, 40 mmol), and DPPP (0.12 g, 0.3 mmol) was bubbled with a gaseous CO for 5 min and then stirred in a CO balloon at 80°C for 4 h. To the reaction mixture was added MeOH (5 ml_). The reaction mixture was stirred for 10 min, diluted with 2N H₂SO₄ (200 ml_), and then extracted with EtOAc. The EtOAc extract was washed with saturated aqueous NaHCO₃, brine, and then dried over Na₂SO₄. Filtration and evaporation of the resultant filtrate yielded a residue, which was purified by flash column chromatography (eluent: EtOAc) to yield compound 1d as a white solid.

Step D: 2-terf-Butoxycarbonylaminoacrylic acid methyl ester

To a suspension of /V-Boc-serine methyl ester (Compound 1e, 2.19 g, 10 mmol) and EDCI (2.01 g, 10.5 mmol) in DCM (70 ml_) was added CuCI (1.04 g, 10.5 mmol). The reaction mixture was stirred at room temperature for 72 h. Upon removal of the solvent, the residue was diluted with EtOAc, washed sequentially with water and brine and then dried over MgSO₄. The crude product was purified by flash column chromatography (eluent: EtOAc:hexane ~1 :4) to yield compound 1f as a colorless oil.

¹H NMR (300 MHz, CDCI₃): δ 1.49 (9H, s), 3.83 (3H, s), 5.73 (1 H, d, J = 1.5 Hz), 6.16 (1 H₁ S), 7.02 (1 H, s).

STEP E: (2)-2-fert-Butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl- phenyl)acrylic acid methyl ester

A flask charged with compound 1d (0.46 g, 2.0 mmol), compound 1f (0.80 g, 4.0 mmol), tri-o-tolylphosphine (0.098 g, 0.32 mmol) and DMF (8 ml_) was purged with N₂(g) 3 times. After the addition of tris(dibenzylideneacetone)dipalladium (0) (0.074 g, 0.08 mmol) and TEA (0.31 ml_, 2.2 mol), the reaction mixture was heated at 110°C for 24 h. At that time, the reaction was quenched by addition of water, and then extracted with EtOAc. The organic phase was washed with 1 N HCI, saturated aqueous NaHCO₃, brine, and dried over MgSO₄. The mixture was concentrated to a residue, which was purified by flash column chromatography (eluent: EtOAc:hexane~1 :1 to EtOAc only) to yield compound 1g as a white solid.

¹H NMR (300 MHz, CD₃OD): δ 1.36 (9H, s), 2.26 (6H, s), 3.83 (3H, s), 7.10 (1 H, s), 7.56 (2H, s); ¹³C NMR (75 MHz, DMSO-d₆): δ 17.6, 25.7, 50.2, 78.7, 124.9, 126.4,

128.3, 131.2, 135.2, 135.5, 152.8, 164.3, 169.6;

MS (ES⁺) (relative intensity): 349.1 (38%)(M+1).

STEP F: (S)-2-ferf-Butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl- phenyl)propionic acid methyl ester

Into a reactor charged with a solution of compound 1g (0.56 g, 1.6 mmol) in degassed MeOH (80 mL) was added [Rh(COd)(H₁R-DIPAMP)J⁺BF₄ ^" under a stream of argon. The reactor was sealed and flushed with H₂, stirred at 6O⁰C under 1000 psi of H₂ for 14 days. The crude product was purified by flash column chromatography (eluent: EtOAc:hexane ~1 :1) to yield compound 1 h as a white solid. ee: >99%; ¹H NMR (300 MHz, CDCI₃): δ 1.36 (9H, s), 2.39 (6H, s), 3.11 (2H, J = 7.2 Hz), 3.65 (3H, s), 4.53-4.56 (1 H, m), 5.12 (1 H, d, J = 8.7 Hz), 5.65 (1 H, br s), 6.09 (1 H, br s), 7.46 (2H, s);

MS(ES⁺) (relative intensity): 250.9 (100) (M-BoC)⁺.

STEP G: (S)-2-tert-Butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl- phenyl)propionic acid

Into an ice-cooled solution of compound "I h (0.22 g, 0.63 mmol) in THF (3.5 ml_) was added an aqueous LiOH solution (1 N, 3.5 ml_) and the reaction mixture stirred at 0°C. Upon completion of the reaction, the reaction mixture was concentrated and the aqueous phase was neutralized with cooled aqueous 1 N HCI at 0°C, and then extracted with EtOAc. The combined extracts were dried over Na₂SO₄ overnight. Filtration and evaporation of the filtrate to dryness yielded compound 1j as a white solid. ¹H NMR (300 MHz, DMSO-cfe): δ 1.30 (9H, s), 2.32 (6H, s), 2.95(1 H, dd,

J= 8.8, 13.9 Hz), 3.10 (1 H, dd, J= 6.2, 14.0 Hz), 4.02-4.12 (1 H, m), 7.18-7.23 (2H, m), 7.48 (2H₁ s), 7.80 (1 H, s);

MS(ES⁺) (relative intensity): 236.9 (6) (M-BoC)⁺.

Example 5

5-((r2-Amino-3-(4-carbamoyl-2.6-dimethyl-phenyl)-propionvn-n-(4-phenyl- 1 H-imidazol-2-yl)-ethvπ-aminol-methyl)-2-methoxy-benzoic acid

STEP A. 2-Methoxy-5-{[1-(4-phenyl-1 W-imidazol-2-yl)-ethylamino]-methyl}- benzoic acid methyl ester

Using the procedures described for Example 4, substituting 5-formyl-2- methoxy-benzoic acid methyl ester (WO 02/22612) for 3,4- dimethoxybenzaldehyde, 2-methoxy-5-{[1 -(4-phenyl-1 H-imidazol-2-yl)- ethylamino]-methyl}-benzoic acid methyl ester was prepared.

STEP B. 5-({[2-ferf-ButoxycarbonylmethyI-3-(4-carbamoyl-2,6-dimethyl- phenyl)-propionyl]-[1 -(4-phenyl-1 H-imidazoI-2-yl)-ethyl]-amino}-methyl)-2- methoxy-benzoic acid methyl ester

Using the procedure of Example 3 for the conversion of Cpd 3d to Cpd 3e, substituting 2-methoxy-5-{[1-(4-phenyl-1 /-/-imidazol-2-yl)-ethylamino]- methylj-benzoic acid methyl ester for Cpd 3d and substituting 2-tert- Butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionic acid for 2- tø/t-Butoxycarbonylamino-3-(4-hydroxy-2,6-dimethyl-phenyl)-propionic acid, Cpd 5a was prepared.

STEP C. 5-({[2-tert-butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl- phenyl)-propionyl]-[1 -(4-phenyl-1 W-imidazol-2-yl)-ethyl]-amino}-methyl)-2- methoxy-benzoic acid

5-({[2-tørf-Butoxycarbonylmethyl-3-(4-carbamoyl-2,6-dimethyl-phenyl)- propionyl]-[1-(4-phenyl-1 H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy- benzoic acid methyl ester was dissolved in an ice-chilled (0-10°C), mixed solvent system of THF (10 ml_) and MeOH (5 ml_). A LiOH H₂O/water suspension (2.48 M; 3.77 ml_) was added dropwise, then the reaction was allowed to warm to room temperature and stirred overnight. The resulting mixture was cooled in an ice bath and the basic solution was neutralized with 2N citric acid until slightly acidic. The mixture was concentrated under reduced pressure to remove the volatile materials, after which time the remaining aqueous phase was extracted with EtOAc (3 x 26 ml_). These combined organic phases were dried over MgSO₄, filtered, and concentrated under reduced pressure to yield a pale yellowish white solid. This crude material was dissolved in a 10% MeOH/CH₂CI₂ solution and adsorbed onto 30 g of silica. The adsorbed material was divided and chromatographed on an ISCO normal phase column over two runs, using a 40 g Redi-Sep column for both runs. The solvent system was a gradient MeOHZCH₂CI₂ system as follows: Initial 100% CH₂CI₂, 98%-92% over 40 min; 90% over 12 min, and then 88% over 13 min. The desired product eluted cleanly between 44-61 min. The desired fractions were combined and concentrated under reduced pressure to yield 5-({[2-terf- butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4- phenyl-1 /-/-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid, Cpd 5b, as a white solid.

STEP D. 5-({[2-Amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1 - (4-phenyl-1 W-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid

A portion of Cpd 5b (0.27g, 0.41 mmol) was dissolved in EtOAc (39 ml_)/THF (5 ml_), filtered, and subsequently treated with gaseous HCI for 15 min. After completion of the HCI addition, the reaction was slowly warmed to room temperature and a solid precipitate formed. After 5 h the reaction appeared >97% complete by LC (@214nm; 2.56 min.). The stirring was continued over 3 d, then the solid was collected and rinsed with a small amount of EtOAc. The resulting solid was dried under high vacuum under refluxing toluene for 2.5 h to yield Cpd 5c as a white solid di-HCI salt.

Example 2

Racemic 2-terf-Butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethvl- phenvD-propionic acid

STEP A: Racemic 2-tert-butoxycarbonylamino-3-(4-carbamoyl-2,6- dimethyl-phenyl)propionic acid methyl ester

To a reactor charged with a solution of compound 1g (0.68 g, 1.95 mmol) in MeOH (80 mL) was added 10% Pd-C (0.5 g). The reactor was connected to a hydrogenator and shaken under 51 psi of H₂ overnight. The mixture was filtered through a pad of Celite and the filtrate was concentrated to dryness to yield compound 2a as a white solid.

The ¹H NMR spectrum was identical to that of (S)-2-tert- butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl-phenyl)propionic acid methyl ester, compound 1 h.

STEP B: Racemic 2-terf-butoxycarbonylamino-3-(4-carbamoyl-2,6- dimethyl-phenyl)propionic acid

Following the procedure described for Example 1 , STEP G (preparation of (S)-2-teAt-Butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl- phenyl)propionic acid), compound 2b – racemic 2-te/?-butoxycarbonylamino-3- (4-carbamoyl-2,6-dimethyl-phenyl)propionic acid – was prepared.

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POLYMORPHS

US8609865

Example 1 Preparation of the zwitterion of 5-({[2-Amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid

A 1 L three-necked round-bottomed flask equipped with a mechanical stirrer, addition funnel and a thermocouple was charged without agitation. 34.2 g of 5-({[2-tert-butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid (see Example 9 of US 2005/0203143), 340 ml of acetone, and 17 ml of 204 mmolar concentrated HCl were combined in the flask. The stirring was started and the resulting slurry formed a clear solution. This solution was heated to 45° C. under vigorous stirring and aged at this temperature for a period of two hours. After the completion, the reaction mass was cooled to ambient temperature and the supernatant was removed by suction. The vessel along with the residue was rinsed with 20 ml of acetone and then removed as previously. 170 ml of water was added and the reaction mass and was aged under stirring until a homogeneus solution resulted. This solution was then added over a period of ˜½ hr to a solution of 90 ml of 1N NaOH and water. The pH was adjusted to 6.5-7.0 accordingly. The resulting slurry was aged for about 2 hrs at ambient temperature, cooled to 10-15° C., aged at that temperature for about 1 hr, and then filtered. The solid was washed with 10 ml water, air-dried for a period of 4 to 5 hrs, and then placed in a vacuum oven at 50-55° C. until the water content was less than 3%.

Example 2 Preparation of the Form α Crystal

The Form α crystal can be prepared by storing the zwitterion of 5-({[2-amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid at 0-25% relative humidity for 3 days. Representative PXRD, TGA, and DSC data are shown in FIGS. 1-3 respectively.

Example 3 Preparation of the Form β crystal

The Form β crystal can be prepared by storing the zwitterion of 5-({[2-amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid at greater than 60% relative humidity for 3 days. Representative PXRD, TGA, and DSC data are shown in FIGS. 1, 4, and 5 respectively.

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SYNTHESIS

US20050203143

Example 9 5-({[2-Amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid

A. 2-Methoxy-5{[1-(4-phenyl-1 H-imidazol-2-yl)-ethylamino]-methyl}-benzoic acid methyl ester.

Using the procedures described for Example 3, substituting 5-formyl-2-methoxy-benzoic acid methyl ester (WO 02/22612) for 3,4-dimethoxybenzaldehyde, 2-methoxy-5-{[1-(4-phenyl-1H-imidazol-2-yl)-ethylamino]-methyl}-benzoic acid methyl ester was prepared.

B. 5-({[2-tert-Butoxycarbonyl methyl-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid methyl ester.

Using the procedure of Example 1 for the conversion of Cpd 1d to Cpd 1e, substituting 2-methoxy-5-{[1-(4-phenyl-1H-imidazol-2-yl)-ethylamino]-methyl}-benzoic acid methyl ester for Cpd 1 d and substituting 2-tert-Butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl-phenyl-propionic acid of Example 8 for 2-tert-Butoxycarbonylamino-3-(4-hydroxy-2,6-dimethyl-phenyl)-propionic acid, Cpd 9a was prepared.

C. 5-({[2-tert-butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[11-(4-phenyl-1H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid.

5-({[2-tert-Butoxycarbonyl methyl-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid methyl ester was dissolved in an ice-chilled (0-10° C.), mixed solvent system of THF (10 mL) and MeOH (5 mL). A LiOH.H₂O/water suspension (2.48 M; 3.77 mL) was added dropwise, then the reaction was allowed to warm to room temperature and stirred overnight. The resulting mixture was cooled in an ice bath and the basic solution was neutralized with 2N citric acid until slightly acidic. The mixture was concentrated under reduced pressure to remove the volatile materials, after which time the remaining aqueous phase was extracted with EtOAc (3×26 mL). These combined organic phases were dried over MgSO₄, filtered, and concentrated under reduced pressure to give 2.26 g (146% of theory) of pale yellowish white solid. This crude material was dissolved in a 10% MeOH/CH₂Cl₂ solution and adsorbed onto 30 g of silica. The adsorbed material was divided and chromatographed on an ISCO normal phase column over two runs, using a 40 g Redi-Sep column for both runs. The solvent system was a gradient MeOH/CH₂Cl₂ system as follows: Initial 100% CH₂Cl₂, 98%-92% over 40 min; 90% over 12 min, and then 88% over 13 min. The desired product eluted cleanly between 44-61 min. The desired fractions were combined and concentrated under reduced pressure to yield 1.74 g (113% of theory) of 5-({[2-tert-butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid, Cpd 9b, as a white solid.

D. 5-({[2-Amino-3-(4-carbamoyl-2,6-dimethyl-phenyl)-propionyl]-[1-(4-phenyl-1H-imidazol-2-yl)-ethyl]-amino}-methyl)-2-methoxy-benzoic acid.

A portion of Cpd 9b (0.27g, 0.41 mmol) was dissolved in EtOAc (39 mL)/THF (5 mL), filtered, and subsequently treated with gaseous HCl for 15 min. After completion of the HCl addition, the reaction was slowly warmed to room temperature and a solid precipitate formed. After 5 h the reaction appeared >97% complete by LC (@214 nm; 2.56 min.). The stirring was continued over 3 d, then the solid was collected and rinsed with a small amount of EtOAc. The resulting solid was dried under high vacuum under refluxing toluene for 2.5 h to yield 0.19 g (71%) of desired Cpd 9c as a white solid di-HCl salt.

Example 8 (S)-2-tert-Butoxycarbonylamino-3-(2,6-dimethyl-4-trifluoromethanesulfonylphenyl)-propionic acid methyl ester

A. (S)-2-tert-Butoxycarbonylamino-3-(2,6-dimethyl-4-trifluoromethanesulfonylphenyl)-propionic acid methyl ester. Into a cool solution of Boc-L-(2,6-diMe)Tyr-OMe (7.0 g, 21.6 mmol; Sources: Chiramer or RSP AminoAcidAnalogues) and N-phenyltrifluoromethanesulfonimide (7.9 g, 22.0 mmol) in dichloromethane (60 mL) was added triethylamine (3.25 mL, 23.3 mmol). The resulting solution was stirred at 0° C. for 1 h and slowly warmed to rt. Upon completion, the reaction was quenched by addition of water. The separated organic phase was washed with 1 N NaOH aqueous solution, water and dried over Na₂SO₄ overnight. After filtration and evaporation, the residue was purified by flash column chromatography (eluent: EtOAc-hexane: 3:7) to give the desired product (9.74 g, 99%) as a clear oil; ¹H NMR (300 MHz, CDCl₃): δ 1.36 (9H, s), 2.39 (6H, s), 3.06 (2H, d, J=7.7 Hz), 3.64 (3H, s), 4.51-4.59 (1H, m), 5.12 (1H, d, J=8.5 Hz), 6.92 (2H, s); MS (ES+) (relative intensity): 355.8 (100) (M−Boc)⁺.

B. (S)₄-(2-tert-Butoxycarbonylamino-2-methoxycarbonylethyl)-3,5-dimethylbenzoic acid. To a suspension of (S)-2-tert-butoxycarbonylamino-3-(2,6-dimethyl-4-trifluoromethanesulfonylphenyl)-propionic acid methyl ester (9.68 g, 21.3 mmol), K₂CO₃ (14.1 g, 0.102 mol), Pd(OAc)₂ (0.48 g, 2.13 mmol) and 1,1′-bis(diphenylphosphino)ferrocene (2.56 g, 4.47 mmol) in DMF (48 mL) was bubbled in gaseous CO for 15 min. The mixture was heated to 60° C. for 8 h with a CO balloon. The cool mixture was partitioned between NaHCO₃ and EtOAc, and filtered. The aqueous layer was separated, acidified with 10% citric acid aqueous solution, extracted with EtOAc, and finally dried over Na₂SO₄. Filtration and concentration of the filtrate resulted in a residue. The residue was recrystallized from EtOAc-hexanes to afford the desired product (7.05 g, 94%); ¹H NMR (300 MHz, CDCl₃): δ 1.36 (9H, s), 2.42 (6H, s), 3.14 (2H, J=7.4 Hz), 3.65 (3H, s), 4.57-4.59 (1H, m), 5.14 (1H, d, J=8.6 Hz), 7.75 (2H, s); MS(ES+) (relative intensity): 251.9 (100) (M−Boc)⁺.

C. (S)-2-tert-Butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethylphenyl)propionic acid methyl ester. Into a stirring solution of (S)-4-(2-tert-butoxycarbonylamino-2-methoxycarbonylethyl)-3,5-dimethyl benzoic acid (3.00 g, 8.54 mmol), PyBOP (6.68 g, 12.8 mmol) and HOBt (1.74 g, 12.8 mmol) in DMF (36 mL) was added DIPEA (5.96 mL, 34.2 mmol) and NH₄Cl (0.92 g, 17.1 mmol). The resulting mixture was stirred at rt for 40 min before being partitioned between aqueous NH₄Cl solution and EtOAc. The separated organic phase was washed sequentially with 2N citric acid aqueous solution, saturated aqueous NaHCO₃ solution, and brine, then dried over Na₂SO₄ overnight. After filtration and concentration, the residue was purified by flash column chromatography (eluent: EtOAc) to give the product. (3.00 g, 100%); ¹H NMR (300 MHz, CDCl₃): δ 1.36 (9H, s), 2.39 (6H, s), 3.11 (2H, J=7.2 Hz), 3.65 (3H, s), 4.53-4.56 (1H, m), 5.12 (1H, d, J=8.7 Hz), 5.65 (1H, brs), 6.09 (1H, br s), 7.46 (2H, s); MS(ES+) (relative intensity): 250.9 (100) (M−Boc)⁺.

D. (S)-2-tert-Butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethylphenyl)propionic acid. Into an ice-cooled solution of methyl ester from Step C (2.99 g, 8.54 mmol) in THF (50 mL) was added an aqueous LiOH solution (1N, 50 mL) and stirred at 0° C. Upon consumption of the starting materials, the organic solvents were removed and the aqueous phase was neutralized with cooled 1N HCl at 0° C., and extracted with EtOAc, and dried over Na₂SO₄ overnight. Filtration and evaporation to dryness led to the title acid (S)-2-tert-butoxycarbonylamino-3-(4-carbamoyl-2,6-dimethylphenyl)propionic acid (2.51 g, 87%); ¹H NMR (300 MHz, DMSO-d₆): δ 1.30 (9H, s), 2.32 (6H, s), 2.95 (1H, dd, J=8.8, 13.9 Hz), 3.10 (1H, dd, J=6.2, 14.0 Hz), 4.02-4.12 (1H, m), 7.18-7.23 (2H, m), 7.48 (2H, s), 7.80 (1H, s); MS(ES+) (relative intensity): 236.9 (6) (M−Boc)⁺.

…………………..

PATENTS

1.WO 2005090315

2..WO 2006099060

3.WO 2009009480

4. WO 2010062590

5.US 2011263868 *

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.

…………………………

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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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.

By: Margaret A. Hamburg, M.D.

We all know that just as every person is different, so too is every disease and every drug. And so we weren’t surprised by the results of a new study published in the Journal of … Continue reading →

http://blogs.fda.gov/fdavoice/index.php/2014/02/why-fda-supports-a-flexible-approach-to-drug-development/?source=govdelivery&utm_medium=email&utm_source=govdelivery

Why FDA Supports a Flexible Approach to Drug Development

IDRAPARINUX

Nonasodium  (2S,3S,4S,5R,6R)-6-[(2R,3R,4S,5R,6R)-6-[(2R,3S,4S,5R,6R)-2-carboxy-4,5-dimethoxy-6-[(2R,3R,4S,5R,6S)-6-methoxy-4,5-disulfooxy-2-(sulfooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-disulfooxy-2-(sulfooxymethyl)oxan-3-yl]oxy-4,5-dimethoxy-3-[(2R,3R,4S,5R,6R)-3,4,5-trimethoxy-6-(sulfooxymethyl)oxan-2-yl]oxyoxane-2-carboxylic acid |

CAS 162610-17-5 (free acid)

SANORG34006, SR-34006, SanOrg 34006, SanOrg-34006, UNII-H84IXP29FN, AC1MJ0N4, Org-34006

Methyl O-2,3,4-tri-O-methyl-6-O-sulfo-alpha-D-glucopyranosyl-(1–4)-O-2,3-di-O-methyl-beta-D-glucopyranuronosyl-(1–4)-O-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1–4)-O-2,3-di-O-methyl-alpha-L-idopyranuronosyl-(1–4)-2,3,6-tri-O-sulfo-alpha-D-glucopyran

Sanofi-Syn(Originator), Organon (Codevelopment), PHASE 3

Methyl O-2,3,4-tri-O-methyl-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-β-D-glucopyranosyluronic acid-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranosyluronic acid-(1→4)-O-α-D-glucopyranose

methyl O-2,3,4-tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranosyl-(1→4)-O-2,3-O-di-methyl-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranoside nonakis sodium salt. [α]D²⁰ = +46.2° (c=1; water). Anomeric protons chemical shifts: 5.43; 5.37; 5.16; 5.09; and 5.09 ppm.

Idraparinux sodium, or methyl O-2,3,4-tri-O-methyl-6-O-sodium sulfonato-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-β-D-glucopyranosyluronate sodium-(1→4)-O-2,3,6-tri-O-sodium sulfonato-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranosyluronate sodium-(1→4)-O-2,3,6-tri-O-sodium sulfonato-α-D-glucopyranose, is a pentasaccharide with antithrombotic activity.

The preparation of idraparinux by sulfatation of a deprotected pentasaccharide is described in Bioorganic & Medicinal Chemistry, 1994, Vol. 2, No. 11, pp. 1267-1280, and also in patent EP 0 529 715 B1.

Idraparinux sodium is an anticoagulant medication in development by Sanofi-Aventis.^[1]

It has a similar chemical structure and the same method of action as fondaparinux, but with an elimination half-life about five to six times longer (an increase from fondaparinux’s 17 hours to approximately 80 hours), which means that the drug should only need to be injected once a week.

As of July 2007, it has completed the Phase III clinical trial AMADEUS.

Idraparinux selectively blocks coagulation factor Xa.^[2]

See Heparin: Mechanism of anticoagulant action for a comparison of the mechanism of heparin, low-molecular-weight heparins, fondaparinux and idraparinux.

Idraparinux sodium is a synthetic pentasaccharide with indirect coagulation factor Xa inhibitor activity. The drug candidate had been in phase III clinical development at Sanofi (formerly known as sanofi-aventis) for the once-weekly long-term treatment and secondary prevention of venous thromboembolic events in patients with pulmonary embolism (PE) and deep vein thrombosis (DVT), as well as for the prevention of thromboembolic complications related to atrial fibrillation (AF).

However, no recent development has been reported for this research. The oligosaccharide is delivered by subcutaneous injection. Unlike other products, idraparinux is administered once weekly rather than daily, thereby increasing patient convenience.

Originally developed under a collaboration between sanofi-sventis and Akzo Nobel’s human healthcare business Organon, all rights to idraparinux were transferred to Sanofi in January 2004 in exchange for revenues based on future sales.

IDRAPARINUX

Several synthetic pentasaccharides have been developed, such as Idraparinux, where all hydroxyl groups are methylated or sulphated, as illustrated below:

Initially, the firm Organon developed a way of synthesis for the preparation of the “active pentasaccharide”. This synthesis, using the 3-0-benzyl-1 ,2-0-isopropylidene-a-D- glucofuranose as substrate (Van Boeckel et al., J. Carbohydr. Chem. 1985, 4, p.293-321 ), comprises more than 50 steps, and the inversion of configuration of the C5 carbon is carried out by the opening of an epoxide. After a step of protection followed by a bromination, the G unit is thus obtained. It is well known that the synthesis of said G unit is very tedious, due to the number of steps for obtaining such unit and the known tendency of L-idose derivatives to exist as furanoses. After being coupled to the H unit, successive steps of protection-deprotection then an oxidation reaction carried out on C6 carbon, lead to the GH disaccharide.

In the preparation of Idraparinux, the synthesis of the disaccharide GH is nearly similar to the above synthesis of early synthetic pentasaccharides. The major innovation lies in the obtaining of disaccharide EF by epimerization of disaccharide GH. The coupling of both disaccharides leads to the tetrasaccharide EFGH, which is further coupled to the D unit for obtaining said pentasaccharide. The preparation of the disaccharide EF from GH allows notably the decrease of the total number of the steps to approximatively 25 (Petitou, M.; Van Boeckel, C.A. Angew. Chem., Int.Ed. 2004, 43, p.31 18-3133).

Hence, all current syntheses of the “active pentasaccharide” comprise a large number of steps and more particularly involves the complex synthesis of key L-iduronic acid derivative (G unit). Indeed, the preparation of the G unit of the “active pentasaccharide” of heparin has always been a limiting step in the synthesis of antithrombotic heparin derivatives.

Thus, there is still a need for a new efficient process of preparation of L-iduronic acid derivative, which would not possess the drawbacks established above and would be compatible with industrial scales. Besides, there is a need for such process which would in addition lead to an improved process of preparation of the “active pentasaccharide” constituting the heparin derivatives.

• Idrabiotaparinux, developed by sanofi-aventis, is the biotinylated pentasaccharide corresponding to the structure depicted below. The pentasaccharide structure of idrabiotaparinux is the same as idraparinux, another antithrombotic agent developed by sanofi-aventis (see structure below). However in idrabiotaparinux, the presence of a biotin hook covalently linked to the first saccharidic unit enables the compound to be neutralized by avidin or streptavidin, as described in the international patent application WO 02/24754 .
• In the EQUINOX trial, which enrolled patients with DVT treated for 6 months with equimolar doses of either idrabiotaparinux or idraparinux, idrabiotaparinux, with the same anti-activated factor X pharmacological activity (hereafter “anti-Xa activity”) as idraparinux, was shown to have a similar efficacy, but, surprisingly, a better safety with less observed bleedings, in particular major bleedings.
• Therefore, the subject-matter of the invention is the use of idrabiotaparinux for the manufacture of a medicament useful for the treatment and secondary prevention of thrombotic pathologies, wherein the use of idrabiotaparinux involves a decrease in the incidence of bleedings during said treatment.
• In other words, the invention relates to the use of idrabiotaparinux as an antithrombotic treatment, wherein said use minimizes the risk of bleedings during the antithrombotic treatment. Indeed, idrabiotaparinux enables to increase the benefit-risk ratio during the antithrombotic treatment.

The L-ioduronic acid methyl ester derivative (XII) is then converted into its D-glucuronic acid methyl ester counterpart (XIII) by epimerization with NaOMe in refluxing MeOH, followed by esterification with MeI and KHCO3 in DMF.

Protection of the ester (XIII) with levulinic acid (IX) by means of DCC and DMAP in dioxane, followed by acetolysis of the anomeric center with sulfuric acid in acetic anhydride furnishes the disaccharide (XIV), which is then saponified with piperidine and subjected to reaction with trichloroacetonitrile and Cs2CO3 in THF to yield the imidate (XV).

Glycosylation of the disaccharide (XII) with the imidate (XV) by means of trimethylsilyl triflate in CH2Cl2, followed by removal of the levulinoyl group by means of hydrazine acetate, furnishes the tetrasaccharide (XVI), which is coupled with the glucosyl trichloroacetimidate (XVIII) by means of trimethylsilyl trifluoromethanesulfonate in CH2Cl2 providing the pentasaccharide (XVII).

Glucosyl imidate (XVIII) is prepared by methylation of 1,6-anhydroglucose (XIX) with MeI and NaH in DMF, followed by acetolysis with Ac2O/TFA to give compound (XX), which is treated with piperidine in THF and finally with trichloroacetonitrile in dichloromethane in the presence of Cs2CO3.

The pentasaccharide (XVII) is deprotected by saponification with LiOH in THF/H2O2, and then hydrogenated over Pd/C in tert-butanol/water to provide a fully deprotected pentamer, which is finally subjected to sulfation with triethylamine sulfur trioxide complex in DMF and converted into the corresponding sodium salt by elution in a Dowex 50 XW4-Na+ or a Mono-Q anion-exchange column.

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Glycosylation of sugar (I) with the idopyranosyl fluoride (II) by means of BF3.Et2O and molecular sieves in dichloromethane gives the disaccharide fragment (III), which is then converted into acetonide (V) by saponification of the ester functions with t-BuOK, followed by reaction with 2,2-dimethoxypropane (IV) in DMF and acidification with p-toluensulfonic acid. Methylation of acetonide (V) with MeI and NaH in DMF/MeOH provides the disaccharide (VI), which is then treated with HOAc to yield the 4′,6′-diol (VII). Selective silylation of the diol (VII) with tert-butyldimethylsilyl chloride (TBDMSCl) in pyridine leads to the 6′-O-TBDMS derivative (VIII), which is condensed with levulinic acid (IX) by means of dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) in dioxane to give the ester (X). Compound (X) is then submitted to simultaneous Jones oxidation and TBDMS removal with CrO3 and H2SO4/H2O in acetone to provide the iduronic acid derivative (XI), which is converted into the key intermediate (XII), first by esterification with MeI and KHCO3 in DMF and then by removal of the 4′-O-levulinoyl protecting group with HOAc and hydrazine hydrate in pyridine.

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US20120041189

Idraparinux sodium, or methyl O-2,3,4-tri-O-methyl-6-O-sodium sulfonato-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-β-D-glucopyranosyluronate sodium-(1→4)-O-2,3,6-tri-O-sodium sulfonato-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranosyluronate sodium-(1→4)-O-2,3,6-tri-O-sodium sulfonato-α-D-glucopyranose, is a pentasaccharide with antithrombotic activity.

The preparation of idraparinux by sulfatation of a deprotected pentasaccharide is described in Bioorganic & Medicinal Chemistry, 1994, Vol. 2, No. 11, pp. 1267-1280, and also in patent EP 0 529 715 B1.

A crystalline form of the pentasaccharide methyl O-2,3,4-tri-O-methyl-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-β-D-glucopyranosyluronic acid-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranosyluronic acid-(1→4)-O-α-D-glucopyranose has now been isolated. This compound in its crystalline form has proven to be very useful for the preparation of idraparinux, since it makes it possible to obtain this product in a particularly interesting chemical yield and with a significant gain in quality, the purity being improved as regards the crude product obtained, as will be detailed hereinbelow. These gains in reaction yield and in purity for the production of idraparinux are considerable advantages from an industrial viewpoint, since improving the robustness of a process is a constant cause for concern, especially in the case of large-scale syntheses.

One subject of the invention is thus the compound methyl O-2,3,4-tri-O-methyl-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-β-D-glucopyranosyluronic acid-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranosyluronic glucopyranose in crystalline form.

Methyl O-2,3,4-tri-O-methyl-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-β-D-glucopyranosyluronic acid-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranosyluronic acid-(1→4)-O-α-D-glucopyranose, referred to hereinbelow as the compound of formula (I), corresponds to the following formula:

The compound of formula (I) in crystalline form according to the invention has a powder X-ray diffractogram whose characteristic lines are approximately at 12.009; 7.703; 7.300; 7.129; 5.838; 4.665; 4.476 and 3.785 angströms (interplanar distances). It also has a melting point of about 203° C. (203° C.±1° C.).

EXAMPLE 1 Preparation of the Compound of Formula (I) in Crystalline Form (Scheme 1)

Methyl O-2,3,4-tri-O-methyl-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-β-D-glucopyranosyluronic acid-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranosyluronic acid-(1→4)-O-α-D-glucopyranose, referred to hereinbelow as the compound of formula (I)

1.1: Preparation of the Compound of Formula (I′)

The compound of formula (I″) is obtained, for example, according to the teaching of patent EP 0 529 715 B1 or of the articles “Bioorg. Med. Chem.” (1994, Vol. 2, No. 11, pp. 1267-1280), “Bioorg. Med. Chem. Letters” (1992, Vol. 2, No. 9, pp. 905-910) or “Magnetic Resonance in Chemistry” (2001, Vol. 39, pp. 288-293). The compound of formula (I″) (5 g, 3.06 mmol) is dissolved in acetonitrile (10 mL). Deionized water (12.2 mL) and aqueous 30% sodium hydroxide solution (4.1 g) are then added. The mixture is heated to 40° C. and maintained at this temperature for 5 hours. The reaction medium is then cooled to 20° C. and acidified to pH 6.25 with aqueous 1N hydrochloric acid solution (about 17.7 g) before extraction with MTBE of certain impurities, the saponified product remaining in the aqueous phase. The residual acetonitrile, contained in the aqueous phase, is then removed by concentration, followed by diluting with deionized water (125 mL). The saponified product is finally precipitated at pH 1.5 by adding aqueous 1N hydrochloric acid solution (about 17.6 g) at 20° C. The suspension is maintained for 4 hours at 20° C. before filtration. The wet solid is finally dried in a vacuum oven at 30° C. to give 2.93 g (93.6%) of compound of formula (I).

NMR (anomeric protons of the saccharide units D, E, F, G, H): 5.79, 5.14, 5.55, 5.92, 4.94 ppm.

1.2 Preparation of the Crude Compound of Formula (I)

The compound of formula (I′) obtained after the preceding step is dissolved in tetrahydrofuran (18 mL). Palladium-on-charcoal (0.3 g) is added. The reaction medium is hydrogenated at 0.3 bar of hydrogen (relative pressure) for 4 hours. After filtering and evaporating, 2.12 g (99%) of the crude compound of formula (I) are obtained.

1.3: Preparation of the Compound of Formula (I) in Crystalline Form Using an Isopropanol/MTBE Mixture

The crude hydrogenated product obtained after the preceding step is dissolved in isopropanol (13 mL) at 65° C., and then crystallized at room temperature. The suspension is then cooled to 40° C., followed by addition of MTBE (13 mL), and is then cooled slowly to 10° C. After maintenance at 10° C. for 2 hours, the crystalline hydrogenated product is filtered off, washed and dried. 1.66 g of the compound of formula (I) in crystalline form are thus obtained, in the form of a cream-white powder. The reaction yield for the production of the compound of formula (I) in crystalline form, from the compound of formula (I′), is 92.5%. When expressed relative to the starting compound (I″), the reaction yield for the production of the compound of formula (I) in crystalline form is 86.6%.

NMR (anomeric protons of the saccharide units D, E, F, G, H) of the compound of formula (I) in crystalline form: 5.77, 5.11, 5.51, 5.84, 5.01 ppm.

1.4: Preparation of the Compound of Formula (I) in Crystalline Form Using Isopropanol

The crude hydrogenated product obtained after step 1.2 is dissolved in isopropanol (5 volumes) at 75° C. The medium is then cooled slowly until crystals appear, according to the known standard techniques for crystallization. The process is performed, for example, by a first step of cooling at 65° C. for 1 hour, and than a second step of cooling to a final temperature of 25° C. over 4 hours or of 5° C. over 6 hours, and finally maintenance at this final temperature for 30 minutes. The suspension is then filtered and rinsed with isopropanol (2×0.1 V) and compound (I) is isolated in the form of white crystals, which appear under a microscope in the form of needles. The ¹H NMR analysis of these crystals is identical to that described after step 1.3 above.

EXAMPLE 4 Preparation of Idraparinux from the Compound of Formula (I) in Crystalline Form (Scheme 2)

The preparation of idraparinux (II) from the compound of formula (I) is summarized in Scheme 2.

The compound of formula (I) in crystalline form, as obtained according to Example 1.3, is dissolved in N,N′-dimethylformamide (6.6 mL) and then heated to 30° C. Under an inert atmosphere, 3.8 g of pyridine-sulfur trioxide complex are added slowly, followed by maintenance at 30° C. for 4 hours. The reaction medium is then poured into aqueous 23.8% sodium hydrogen carbonate solution (16.3 g) maintained at a maximum of 25° C., to obtain the compound of formula (II). The reaction medium is kept stirring for hours. The solution of sulfated product is then poured onto an MTBE/isopropanol/ethanol mixture (171 mL/70 mL/70 mL). Precipitation of the product is observed, and, after filtering off, washing and drying the cake, 4.99 g (96.8%) of compound of formula (II) are obtained, and are then purified by anion-exchange chromatography according to the usual techniques.

NMR (anomeric protons of the saccharide units D, E, F, G, H) of the compound of formula (II): 5.48, 4.68, 5.44, 5.08, 5.18 ppm.

It thus appears that the process according to the invention makes it possible to obtain idraparinux (compound of formula (II)) in a chemical yield of about 84% (precisely 83.8% according to the protocols described above) starting from the compound of formula (I″), i.e. a gain in yield of about 30% relative to the process described in patent EP 0 529 715 B1.

………………..

EP0529715A1

methyl O-2,3,4-tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranosyl-(1→4)-O-2,3-O-di-methyl-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranoside nonakis sodium salt. [α]_D²⁰ = +46.2° (c=1; water). Anomeric protons chemical shifts: 5.43; 5.37; 5.16; 5.09; and 5.09 ppm.

WAS PREPARED AS PER

Example 3

methyl O-4-O-(4-sulfoaminophenyl)-2,3,6-tri-O-sulfo-α-D-glucopyranosyl-(1→4)-O-3-O-methyl-2-O-sulfo-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranoside nonakis sodium salt.  

NOTE THIS IS ANALOGOUS PROCEDURE AND NOT SIMILAR

• Methyl O-4-O-(4-nitrophenyl)-6-O-acetyl-2,3-O-di-phenylmethyl-α-D-glucopyranosyl-(1→4)-O-(methyl 3-O-methyl-2-O-acetyl-α-L-idopyranosyluronate)-(1→4)-O-2,3,6-tri-O-acetyl-α-D-glucopyranoside (100 mg, 0.09 mmol), obtained by the known imidate coupling of the trichloroacetimidate of O-4-O-(4-nitrophenyl)-6-O-acetyl-2,3-O-di-phenylmethyl-α-D-glucopyranoside and methyl O-(methyl 3-O-methyl-2-O-acetyl-α-L-idopyranosyluronate)-(1→4)-O- 2,3,6-tri-O-acetyl-α-D-glucopyranoside, was dissolved in tetrahydrofuran (9 ml) and cooled to -5 °C. At this temperature a 30% aq. solution of hydrogen peroxide (4.5 ml) was added to the reaction mixture, and after 10 min a 1.25 M lithium hydroxide solution (4.7 ml) was added. The mixture was stirred for 1 h at -5 °C, after which time the temperature was raised to 0 °C and the mixture was stirred overnight. The reaction mixture was acidified with 6N hydrogen chloride at 0 °C to pH 1.5, after which the saponified compound was extracted with ethyl acetate. The organic layers were pooled, dried over magnesium sulfate, and evaporated to give 63 mg (84%) of methyl O-4-O-(4-nitrophenyl)-2,3-O-di-phenylmethy1-α-D-glucopyranosyl-(1→4)-O-3-O-methyl-α-L-idopyranuronosyl-(1→4)-O-α-D-glucopyranoside, which was dissolved in methanol (8 ml). 10% Pd on charcoal (63 mg) was added and the mixture hydrogenolyzed overnight. After filtration and evaporation 27 mg (50%) of methyl O-4-O-(4-aminophenyl)-α-D-glucopyranosyl-(1→4)-O-3-O-methyl-α-L-idopyranuronosyl-(1→4)-O-α-D-glucopyranoside were obtained.
  13 mg of methyl O-4-O-(4-aminophenyl)-O-α-D-glucopyranosyl-(1→4)-O-3-O-methyl-α-L-idopyranuronosyl-(1→4)-O-α-D-glucopyranoside were dissolved in 2 ml of dry N,N-dimethylformamide, and under an atmosphere of nitrogen 148 mg of triethylamine sulfurtrioxide complex were added. The mixture was stirred overnight at 50 °C, after which an aq. solution of sodium hydrogen carbonate was added under ice cooling. The mixture was stirred for 1 h at room temperature, concentrated to a small volume and desalted on a Sephadex G-10 column with water. The crude product obtained was purified by HPLC using a Mono-Q anion exchange column to give 11 mg (37%) of methyl O-4-O-(4-sulfoaminophenyl)-2,3,6-tri-O-sulfo-α-D-glucopyranosyl-(1→4)-O-3-O-methyl-2-O-sulfo-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranoside nonakis sodium salt. [α]_D²⁰ = +52.2° (c=0.67; water). Anomeric protons chemical shifts: 5.5; 5.17; and 5.15 ppm.

………………………………..

BMCL Volume 19, Issue 14, 15 July 2009, Pages 3875–3879

http://www.sciencedirect.com/science/article/pii/S0960894X0900482X

Final elaboration of the pentasaccharide 1. Reagents and conditions: (a) TMSOTf, Et₂O, 4 Å MS, rt, 66% (28α), 15% (28β); (b) CAN, CH₃CN, toluene, H₂O, rt, 72%; (c) CCl₃CN, DBU, CH₂Cl₂, rt, 98%; (d) TMSOTf, 4 Å MS, CH₂Cl₂, rt, 51% (73% based on recovery of 4); (e) Pd/C (10%), H₂, t-BuOH, H₂O, rt; (f) SO₃·Et₃N, DMF, 50 °C, 93% (2 steps).

The final elaboration of the pentasaccharide 1 was illustrated in IN ABOVE SCHEME Coupling of the glucopyranosyl trichloroacetimidate 6 with disaccharide acceptor 5 in the presence of trimethylsilyl trifluoromethylsulfonate and powdered 4 Å molecular sieves at room temperature in diethyl ether afforded the desired α-coupled trisaccharide 28α in a yield of 66%, together with 15% of the separable β-coupled product 28β. The anomeric 4-methoxyphenyl group in trisaccharide 28α was removed with CAN, and the resulting lactol was readily converted into the trisaccharide trichloroacetimidate 3. Coupling of donor 3 with the disaccharide acceptor 4 in the presence of trimethylsilyl trifluoromethylsulfonate and powdered 4 Å molecular sieves at room temperature in dichloromethane afforded the fully protected pentasaccharide 2 in 51% yield (73% based on recovery of 4). Finally, pentasaccharide 2 was subject to hydrogenolysis of the benzyl protecting groups. The highly polar product without purification was O-sulfated directly with triethylamine-sulfur trioxide complex to afford the sulfated pentasaccharide 1  in an excellent yield of 93% (for two steps).

Summarizing, the potent anti-thromboembolic pentasaccharide Idraparinux (1) was synthesized in total 51 steps and in 4% overall yield from d-glucose and methyl α-d-glucopyranoside.¹⁸ The synthetic route is convergent with a linear sequence of 27 steps, and the transformations are scalable. The 4-methoxyphenol glycoside intermediates are easy to be purified by crystallization.

Compound 1:  54.2 (c 1.0, H₂O);

¹H NMR (400 MHz, D₂O) δ 3.27 (t, J = 8.4 Hz, 1H), 3.30–3.38 (m, 2H), 3.47 (s, 3H), 3.53 (s, 3H), 3.56 (s, 6H), 3.58 (s, 3H), 3.62 (s, 3H), 3.63 (s, 3H), 3.64 (s, 6H), 3.75 (d, J = 10.0 Hz, 1H), 3.83–3.97 (m, 4H), 3.98 (t, J = 8.8 Hz, 1H), 4.06–4.18 (m, 3H), 4.19–4.45 (m, 8H), 4.56 (br t, J = 9.6 Hz, 1H), 4.65 (t, J = 9.2 Hz, 1H), 4.66 (d, J = 7.6 Hz, 1H), 5.00 (br s, 1H), 5.11 (br s, 1H), 5.17 (d, J = 3.6 Hz, 1H), 5.43 (d,J = 3.2 Hz, 1H), 5.47 (d, J = 4.0 Hz, 1H);

ESI-MS m/z 774.1 [M−8Na+6H]²⁻, 763.0; [M−9Na+7H]²⁻, 508.5 [M−9Na+6H]³⁻.

………………….

WO2013050497A1

The process of preparation of Idraparinux having the following formula:

may comprise the following steps :

1 ) preparation a compound of formula (IXB)

(IXB) wherein R_a is methyl, R_b is methyl, R_c is methyl, T-i is benzyl, T₂ is benzyl, T₃ is benzyl and T is methyl, by the process according to the invention;

2) epimerisation of the disaccharide (IXB) so as to form disaccharide D of formula :

3) protection of the 4′-OH of D with a levulinoyl ester;

4) acetolysis of the disaccharide resulting from step 3), followed by preparation of the corresponding imidate;

5) coupling the disaccharide imidate resulting from step 4) with (IXB) obtainable by the process of the invention, wherein R_a is methyl, R_b is methyl, R_c is methyl, T-i is benzyl, T₂ is benzyl, T₃ is benzyl and T is methyl, to obtain a tetrasaccharide;

6) coupling the fully protected tetrasaccharide with a monosaccharide glycosyl imidate;

7) deprotection of the protecting groups by the successive saponification and hydrogenolysis;

8) sulfation of the hydroxyl groups.

In one embodiment, the present invention concerns a process of preparation of Idraparinux:

said process comprising the following steps:

preparation of a compound of formula (VI) such as defined above, from a compound of formula (V) such as defined above; preparation of a compound of formula (VII) such as defined above, from a compound of formula (VI) such as defined above;

preparation of a compound of formula (VIII) such as defined above, from a compound of formula (VII) such as defined above;

- preparation of a compound of formula (IX) such as defined above, from a compound of formula (VIII) such as defined above;

wherein in compounds of formulae (V), (VI), (VII), (VIII) and (IX), R-i , R₂, R3 and X are as defined above, R_a is methyl, R_b is methyl, R_c is methyl, R_d is methyl and R’ is the monosaccharide of formula :

wherein T-i is benzyl, T₂ is benzyl, T₃ is benzyl and T is methyl. The inventors advantageously found that the process of preparation of Idraparinux comprising the decarboxylation/intramolecular cyclisation tandem reaction, which allows the inversion of configuration of C5 carbon of the compound of formula (VI), is more efficient than the processes previously described in the literature. Indeed, the process according to the invention allows advantageously a significant decrease of the number of steps and thus an improvement of the overall yield. Thus, the process of preparation of Idraparinux may be carried out in industrial scales. The inventors found an efficient process of preparation of Idraparinux.

According to another object, the present invention concerns the use of compounds of formulae (V), (VI), (VI I), (VIII) and (IX), as intermediates for the preparation of Idraparinux. In particular, the present invention concerns the use of a compounds of formulae (VB), (VI B), (VI IB), (VI I IB) and (IXB), as intermediates for the preparation of Idraparinux The invention is further illustrated but not restricted by the description in the following examples. Example 1 :

Preparation of Methyl-4,6-0-benzylidene-a-D-glucopyranoside (la)

CHC1₃)

T_f = 166-167°C (litt. 165-166°C) To a solution of benzaldehyde (400 mL, 3.94 mol, 5.9 eq.) was added zinc chloride (100.3 g, 0.74 mol, 1 .1 eq.) under vigorous stirring. After homogenization of the solution methyl- a-D-glucopyranoside (129.6 g, 0.67 mol, 1.0 eq.) was added portionwise. After 16 hours stirring at room temperature the reaction mixture was diluted with diethyl ether (100 mL). The mixture was then poured dropwise and under vigorous stirring in a solution containing ice water (1 .5 L) and hexane (350 mL). The precipitate was filtered, washed with diethyl ether (3 x 300 mL) and dried under vacuum over KOH. The product was then recrystallised from CH₂CI₂ (720 mL) and washed with a Et₂0/CH₂CI₂ solution (75:25, 2 x 200 mL). The filtrate was repeatedly recrystallised five times from CH₂CI₂ to afford compound la as white crystals (136.97 g, 0.49 mol, 72%).

¹H NMR (CDCI3, 250 MHz): δ 2.35 (d, J_CH-O_H = 9.2 Hz, 1 H, OH), 2.83 (d, J_CH-O_H = 2.2 Hz, 1 H, OH), 3.46 (s, 3H, -OCH₃), 3.43-3.46 (m, 1 H, H-4), 3.63 (td, J_CH-_OH = ^_2,3 = 9.2 Hz, J_{1 2} = 3.9 Hz, 1 H, H-2), 3.70-3.81 (m, 2H, H-5, H-6), 3.93 (td, J = 9.2 Hz, J_CH-_OH = 2.2 Hz, 1 H, H- 3), 4.29 (m, 1 H, H-6 ), 4.79 (d, J_1i2 = 3.9 Hz, 1 H, H-1 ), 5.54 (s, 1 H, Ph-CH), 7.35-7.38 (m, 3H, H_Ar), 7.47-7.51 (m, 2H, H_Ar).

¹³C NMR (CDCI₃, 62.9 MHz): δ 55.6 (-OCH₃), 62.5 (C-5), 69.0 (C-6), 71.1 (C-3), 72.9 (C- 2), 81 .0 (C-4), 99.9 (C-1 ), 102.0 (Ph-CH), 126.4, 128.5, 129.4, 137.1 (6xC_Ar). IR (film) v (cm^“1): 3369 (O-H). Preparation of Methyl-2,3-di-0-methyl-4,6-0-benzylidene-a-D-glucopyranoside (Ma)

1₃)

°C)

To a solution of compound la (47.60 g, 0.17 mol, 1.0 eq.) in anhydrous THF (750 mL) under an argon atmosphere and cooled to 0°C, was added portionwise sodium hydride (60%, 16.93 g, 0.42 mol, 2.5 eq.). After 20 minutes methyl iodide was added dropwise (30 mL, 0.48 mol, 2.8 eq.) and the reaction mixture was allowed to reach room temperature. After 16 hours, methanol was added portionwise (75 mL) and the solution was stirred for another 15 minutes before being concentrated. The resulting residue was dissolved in EtOAc (400 mL) and washed with water (2 x 250 mL). The organic layer was dried (MgS0₄), filtered and concentrated. The resulting solid was dissolved in diethyl ether (1000 mL), hexane was added (400 mL) and the solvent was partially evaporated at low temperature. The crystals obtained were washed with hexane and the filtrate once again partially evaporated, filtered and the precipitate washed with hexane. The combined precipitates afforded compound Ma as white crystals (46.10 g, 0.15 mol, 88%).

¹H NMR (CDCI₃, 250 MHz): δ 3.30 (dd, J_2-3 = 9.1 Hz, J_1-2 = 3.7 Hz, 1 H, H-2), 3.44 (s, 3H, – OCH₃), 3.49-3.87 (m, 4H, H-4, H-5, H-6, H-3), 3.55 (s, 3H, -OCH₃), 3.64 (s, 3H, -OCH₃), 4.28 (dd, J_6-6. = 9.1 Hz, J_5-6 = 3.7 Hz, 1 H, H-6 ), 4.85 (d, J_1-2 = 3.7 Hz, 1 H, H-1 ), 5.54 (s, 1 H, Ph-CH), 7.36-7.41 (m, 3H, H_Ar), 7.48-7.52 (m, 2H, H_Ar).

¹³C NMR (CDCI₃, 62.9 MHz): δ 55.4, 59.5, 61.1 (3x-OCH₃), 62.3 (C-5), 69.1 (C-6), 79.9 (C-3), 81 .5 (C-4), 82.2 (C-2), 98.5 (C-1 ), 101.4 (Ph-CH), 126.2, 128.3, 129.0, 137.4 (6xC_Ar).

Preparation of Methyl-2,3-di-0-methyl-a-D-glucopyranoside (Ilia)

1₃)

To a suspension of compound Ma (10.33 g, 33.29 mmol, 1.0 eq.) in methanol (150 mL) was added para-toluenesulfonic acid monohydrate (322 mg, 1 .69 mmol, 0.05 eq.). After 4 hours stirring at room temperature, sodium carbonate (300 mg) was added and the reaction mixture was stirred an additional 15 minutes before filtration through a pad of Celite^®. Then the filtrate was concentrated and the residue obtained was dissolved in a mixture of distilled water/diethyl ether (3:1 , 150 mL). The organic layer was extracted with water (2 x 50 mL) then the combined aqueous phases were concentrated and dried one night under vacuum over KOH. The resulting residue was recrystallised from toluene using petroleum ether as a co-solvent. The crystals obtained were washed with hexane and dried under vacuum over KOH to obtain compound Ilia as white crystals (6.63 g, 29.83 mmol, 90%).

¹H NMR (DMSO, 400 MHz): δ 3.03 (dd, J_2-3 = 9.3 Hz, J_1-2 = 3.5 Hz, 1 H, H-2), 3.12-3.20 (m, 2H, H-3, H-4), 3.27 (s, 3H, -OCH₃), 3.32 (s, 3H, -OCH₃), 3.30-3.33 (m, 1 H, H-5), 3.44 (s, 3H, -OCH3), 3.40-3.46 (m, 1 H, H-6), 3.62 (ddd, J_6-6‘ = 1 1.6 Hz, J_CH-OH = 5.7 Hz, J_5-6 = 1 ,9 Hz, 1 H, H-6′), 4.52 (t, J = 5.7 Hz, 1 H, OH), 4.78 (d, Ji_-2 = 3.5 Hz, 1 H, H-1 ), 5.09 (d,

¹³C NMR (DMSO, 100.6 MHz): δ 54.1 , 57.4, 60.0 (3x-OCH₃), 60.6 (C-6), 69.5 (C-3), 72.5 (C-5), 80.9 (C-2), 82.8 (C-4), 96.4 (C-1 ).

IR (film) v (cm^“1): 3419 (O-H).

Preparation of Methyl methyl-2,3-di-0-methyl-a-D-glucopyranosiduronate (IVa)

C1₃)

To a solution of compound Ilia (500 mg, 2.25 mmol, 1.0 eq.) in distilled water (15 mL) were successively added NaBr (50 mg, 0.49 mmol, 0.2 eq.) and TEMPO (7 mg, 0.05 mmol, 0.02 eq.). The reaction mixture was cooled with the aid of an ice bath then a solution of NaOCI (13% v/v, 5.2 mL, 9.1 mmol, 4.0 eq.) was added. After 5 hours stirring at 0°C ethanol was added (96% v/v, 8 mL), then the pH was reduced to 2-3 by addition of HCI (10 % v/v). The solvent was evaporated and the residue obtained was suspended in methanol, filtered in order to remove the remaining salts and washed several times with dichloromethane and methanol. The filtrate was concentrated then dissolved, under an argon atmosphere, in dry methanol (40 mL). para-toluenesulfonic acid (85 mg, 0.45 mmol, 0.2 eq.) was added then the reaction mixture was heated under reflux overnight. The solvent was evaporated and the residue obtained was dissolved in EtOAc (60 mL). The organic layer was washed with a 5% aqueous NaHC0₃ solution (2 ^χ 20 mL) and with brine (1 ^χ 20 mL). The aqueous phase was extracted with dichloromethane (3 ^χ 20 mL). The combined organics were dried (MgS0₄), filtered and evaporated. Column chromatography (hexane/ethyl acetate 50:50) gave compound IVa as a colourless oil (503 mg, 2.00 mmol, 89%).

¹H NMR (CDCIs, 400 MHz): δ 3.10 (d, J_CH-OH = 3.0 Hz, 1 H, OH), 3.26 (dd, J_2-3 = 9.3 Hz, J_{1 -2} = 3.4 Hz, 1 H, H-2), 3.47 (s, 3H, -OCH₃), 3.49-3.52 (m, 1 H, H-3), 3.50 (s, 3H, -OCH₃), 3.62 (s, 3H, -OCH₃), 3.74 (td, J = 9.5 Hz, J_CH-OH = 3.0 Hz, 1 H, H-4), 3.82 (s, 3H, -OCH₃), 4.14 (d, J4.5 = 9.6 Hz, 1 H, H-5), 4.91 (d, Ji_-2 = 3.4 Hz, 1 H, H-1 ).

¹³C NMR (CDCI₃, 100.6 MHz): δ 52.9, 56.0, 59.1 , 61 .3 (4x-OCH₃), 70.6 (C-5), 71.7 (C-4), 80.9 (C-2), 81.8 (C-3), 98.1 (C-1 ), 170.9 (C=0).

IR (film) v (cm^“1): 3475 (O-H), 1750 (C=0).

Preparation of Methyl methyl^-O-il’-ethoxy^’-propyn-l’-ylJ^.S-di-O-methyl-a-D- glucopyranosiduronate (Va)

To a solution of compound IVa (4.56 g, 18.21 mmol, 1.0 eq.) in chloroform (stabilised with amylene, 200 mL) were added, under an argon atmosphere, P₂0₅ (5.31 g, 36.29 mmol, 2.0 eq.) and propargylaldehyde diethylacetal (5.2 mL, 36.27 mmol, 2.0 eq.), then the reaction mixture was heated at 60°C. After 4 hours stirring, the reaction mixture was filtered through a pad of Celite^® then the solvent was removed under vacuum. The crude mixture was suspended in EtOAc (300 mL), washed with a 5% NaHC0₃ aqueous solution (1 x 30 mL) and brine (1 x 30 ml_). The organic layer was dried (MgS0₄), filtered, and evaporated. Column chromatography (gradient hexane/ethyl acetate 80:20 – 20:80) afforded compound Va as a colourless oil (4.07 g, 12.24 mmol, 67%) in a diastereomeric mixture (64:36) (the relative composition of the mixture was determined by ¹H NMR from integrations of protons EtO-CH), along with some unreacted compound IVa (1.17 g, 4.68 mmol, 26%).

¹H NMR (CDCI₃, 400 MHz): δ 1.18-1.25 (m, 3H, -OCH₂CH₃) (diastereomeric mixture), 2.56 (m, 1 H, H-C≡C-) (mixture), 3.26-3.31 (m, 1 H, H-2) (mixture), 3.43 (s, 3H, -OCH₃) (major), 3.44 (s, 3H, -OCH₃) (minor), 3.50 (s, 3H, -OCH₃) (mixture), 3.59 (s, 3H, -OCH₃) (minor), 3.62 (s, 3H, -OCH₃) (major), 3.47-3.62 (m, 2H, H-3, -OCH_aH_bCH₃) (mixture), 3.65-3.73 (m, 1 H, -OCH_aH_bCH₃) (mixture), 3.78 (s, 3H, -OCH₃) (major), 3.80 (s, 3H, -OCH₃) (minor), 3.78-3.86 (m, 1 H, H-4) (mixture), 4.15 (d, J_4-5 = 10.0 Hz, 1 H, H-5) (major), 4.18 (d, J_4-5 = 10.0 Hz, 1 H, H-5) (minor), 4.86-4.88 (m, 1 H, H-1 ) (mixture), 5.35 (d, J = 1.7 Hz, 1 H, EtO- CH) (minor), 5.58 (d, J = 1.7 Hz, 1 H, EtO-CH) (major).

¹³C NMR (CDCI₃, 100.6 MHz): δ 15.0 (-OCH₂CH₃) (mixture), 52.6 (-OCH₃) (major), 52.7 (- OCH₃) (minor), 55.8 (-OCH₃) (mixture), 59.2 (-OCH₃) (major), 59.3 (-OCH₃) (minor), 60.4 (-OCH₂CH₃) (major), 61 .3 (-OCH₂CH₃) (minor), 61.4 (-OCH₃) (mixture), 70.1 (C-5) (minor), 70.2 (C-5) (major), 74.0 (H-C≡C-) (major), 74.2 (H-C≡C-) (minor), 76.4 (C-4) (minor), 76.7 (C-4) (major), 78.6 (H-C≡C-) (minor), 78.9 (H-C≡C-) (major), 81 .5 (C-2) (major), 81 .8 (C-2) (minor), 81.9 (C-3) (minor), 82.9 (C-3) (major), 92.6 (EtO-CH) (mixture), 97.9 (C-1 ) (minor), 98.0 (C-1 ) (major), 169.6 (C=0) (major), 169.9 (C=0) (minor). Elemental analysis: Calculated: C: 54.21 ; H: 7.28. Found: C: 54.17 ; H: 7.13.

ESI-MS (pos. mode): m/z = 355 [M+Na]⁺.

IR (film) v (cm^“1): 1752 (C=0), 3266 (≡C-H). Preparation of 4-0-(1′-ethoxy-2′-propyn-1^,-yl)-1 ,2,3-tri-0-methyl-a-D-gluco- pyranosiduronic acid (Via)

To a solution of compound Va (1.12 g, 3.37 mmol, 1.0 eq.) in EtOH/H₂0 (3:1 , 100 mL) was added sodium hydroxide (156 mg, 3.90 mmol, 1 .3 eq.). After 5 hours stirring at room temperature the solvent was evaporated. The residue obtained was dissolved in water (50 mL). The pH of the aqueous layer was reduced to 2-3 with a 5% citric acid aqueous solution, then the layer was saturated with sodium chloride before extraction with dichloromethane (10 x 20 mL). If necessary the pH was adjusted by addition of more citric acid aqueous solution. The combined organics were dried (MgS0₄), filtered and removed under vacuum. Compound Via was obtained without further purification as a colourless oil (1.020 g, 3.20 mmol, 95%), in a mixture of diastereomers (75:25) (the relative composition of the mixture was determined by ¹H NMR from integrations of protons EtO-CH).

¹H NMR (CDCIs, 400 MHz): δ 1.16-1.24 (m, 3H, -OCH₂CH₃) (diastereomeric mixture), 2.59 (d, J = 1 .6 Hz, 1 H, H-C≡C-) (major), 2.62 (s I, 1 H, H-C≡C-) (minor), 3.25-3.33 (m, 1 H, H- 2), 3.44 (s, 3H, -OCH₃) (mixture), 3.51 (s, 3H, -OCH₃) (mixture), 3.62 (s, 3H, -OCH₃) (mixture), 3.54-3.62 (m, 2H, H-3, -OCH_aH_bCH₃) (mixture), 3.68-3.77 (m, 1 H, -OCH_aH_bCH₃) (mixture), 3.81-3.87 (m, 1 H, H-4) (mixture), 4.13-4.18 (m, 1 H, H-5) (mixture), 4.88-4.90 (m, 1 H, H-1 ), 5.45 (s I, 1 H, EtO-CH) (minor), 5.63 (d, J = 1.6 Hz, 1 H, EtO-CH) (major).

¹³C NMR (CDCI₃, 100.6 MHz): δ 14.9 (-OCH₂CH₃) (mixture), 55.9 (-OCH₃) (mixture), 59.2 (-OCH₃) (minor), 59.3 (-OCH₃) (major), 60.7 (-OCH₂CH₃) (mixture), 61 .2 (-OCH₃) (minor), 61.3 (-OCH₃) (major), 70.1 (C-5) (mixture), 74.3 (H-OC-) (major), 74.8 (H-OC-) (minor), 75.7 (C-4) (minor), 76.4 (C-4) (major), 78.5 (H-C≡C-) (minor), 78.8 (H-C≡C-) (major), 81.4 (C-2) (major), 81 .7 (C-2) (minor), 81 .8 (C-3) (minor), 82.9 (C-3) (major), 92.5 (EtO-CH) (mixture), 97.8 (C-1 ) (minor), 98.0 (C-1 ) (major), 173.8 (C=0) (major), 174.0 (C=0) (minor).

ESI-MS (pos. mode): m/z = 341 [M+Na]⁺. IR (film) v (cm^“1): 1751 (C=0), 3268 (≡C-H).

Preparation of Methyl-4,7-anhydro-6-deoxy-6-methylene-7-ethoxy-2,3-di-0-methyl- a-L-/^‘d -heptopyranoside (Vila)

To a solution of compound Via (1.89 g, 5.92 mmol, 1 .0 eq.) in anhydrous THF (40 mL) and cooled to 0°C, were added IBCF (0.84 mL, 6.48 mmol, 1.1 eq.) and N- methylmorpholine (0.72 mL, 6.55 mmol, 1.1 eq.). After 20 minutes stirring the flask was covered with aluminium foil, 2-mercaptopyridine /V-oxide sodium salt (1 .77 g, 1 1.80 mmol, 2.0 eq.) was added and the reaction mixture was stirred at ambient temperature. After 2 hours, anhydrous THF (80 mL) then ie f-butylthiol (0.28 mL, 2.61 mmol, 1.6 eq.) were added. The aluminium foil was removed and the reaction mixture was irradiated and heated 30 minutes with a UV lamp (300W). The thiol excess was neutralized with a NaOCI aqueous solution (13% v/v, 10 mL). The reaction mixture was concentrated then dissolved in EtOAc (100 mL), washed successively with a 5% NaHC0₃ aqueous solution (2 x 15 mL), a 5 % citric acid aqueous solution (1 x 15 mL) and brine (1 x 25 mL), then the aqueous layer was extracted with dichloromethane (2 x 20 mL). The combined organics were dried (MgS0₄), filtered and concentrated. Column chromatography (gradient dichloromethane/ethyl acetate 95:5 – 75:25) afforded compound Vila as a colourless oil (218 mg, 0.79 mmol, 48%), in a mixture of diastereomers (67:33) (the relative composition of the mixture was determined by ¹H NMR from integrations of protons H-2).

¹H NMR (CDCIs, 400 MHz): δ 1.23 (t, J = 7.1 Hz, 3H, -OCH₂CH₃) (diastereomeric mixture), 3.13 (dd, J_2-3 = 9.6 Hz, J_1-2 = 3.0 Hz, 1 H, H-2) (minor), 3.30 (dd, J_2-3 = 5.0 Hz, J_1-2 = 1.6 Hz, 1 H, H-2) (major), 3.41 (s, 3H, -OCH₃) (minor), 3.47 (s, 3H, -OCH₃) (major), 3.50 (s, 3H, -OCH₃) (mixture), 3.53 (s, 3H, -OCH₃) (major), 3.55-3.61 (m, 1 H, -OCH_aH_bCH₃) (mixture), 3.72 (dd, J_2-3 = 5.0 Hz, J_3-4 = 2.8 Hz, 1 H, H-3) (major), 3.78-3.90 (m, 1 H, – OCH_aH_bCH₃) (mixture), 3.93-4.07 (m, 2H, H-3, H-4) (mixture), 4.59 (d I, J_4-5 = 4.0 Hz, 1 H, H-5) (major), 4.62 (d, J_1-2 = 1.7 Hz, 1 H, H-1 ) (major), (td, J_4-5 = 7.9 Hz, J = 2.6 Hz, 1 H, H- 5), (minor), 4.79 (d, J_1-2 = 3.0 Hz, 1 H, H-1 ) (minor), 5.35-5.57 (m, 3H, H-7, -C=CH₂) (mixture). ¹³C NMR (CDCI3, 100.6 MHz): δ 15.3 (-OCH₂CH₃) (minor), 15.4 (-OCH₂CH₃) (major), 56.5 (-OCH3) (minor), 56.8 (-OCH₃) (major), 58.6 (-OCH₃) (major), 59.1 (-OCH₃) (minor), 59.9 (- OCH3) (major), 60.3 (-OCH₃) (minor), 63.3 (-OCH₂CH₃) (minor), 63.8 (-OCH₂CH₃) (major), 74.2 (C-5) (minor), 74.7 (C-5) (major), 76.4 (C-3) (major), 77.0 (C-4) (major), 77.7 (C-2) (major), 79.0 (C-3) (minor), 79.7 (C-4) (minor), 80.1 (C-2) (minor), 99.3 (C-1 ) (major), 99.5 (C-1 ) (minor), 102.2 (C-7) (major), 103.0 (C-7) (minor), 1 1 1.6 (-C=CH₂) (minor), 1 15.2 (- C=CH₂) (major), 147.4 (C-6) (minor), 148.1 (C-6) (major).

ESI-MS (pos. mode): m/z = 297 [M+Na]⁺.

Preparation of Methyl-4,7-anhydro-7-ethoxy-2,3-di-0-methyl-a-L-/ o-hepto- pyranosid-6-ulose (Villa)

Through a solution of compound Vila (449 mg, 1 .64 mmol, 1.0 eq.) in anhydrous dichloromethane (10 ml_), under an argon atmosphere and cooled to -78°C, was bubbled ozone (0.2 L/min, 1 10 V). When the solution had turned dark blue, oxygen was bubbled through in order to remove the excess ozone. When the solution became colorless dimethylsulfide (5 drops) was added and the solution was brought to room temperature. After 1 h15 the reaction mixture was concentrated. Column chromatography (gradient dichloromethane/ethyl acetate 95:5 – 80:20) afforded compound Villa as a white solid (364 mg, 1.32 mmol, 80%), in a mixture of diastereomers (79:21 ) (the relative composition of the mixture was determined by ¹H NMR from integrations of protons H-2).

¹H NMR (CDCI₃, 400 MHz): δ 1.24-1 .28 (m, 3H, -OCH₂CH₃) (diastereomeric mixture), 3.10 (dd, J_2-3 = 10.2 Hz, J_1-2 = 2.9 Hz, 1 H, H-2) (minor), 3.17 (dd, J_2-3 = 9.4 Hz, J_1-2 = 2.8 Hz, 1 H, H-2) (major), 3.38 (s, 3H, -OCH₃) (major), 3.42 (s, 3H, -OCH₃) (minor), 3.50 (s, 3H, – OCH₃) (mixture), 3.63 (s, 3H, -OCH₃) (major), 3.66 (s, 3H, -OCH₃) (minor), 3.48-3.73 (m, 3H, H-3 major, -OCH_aH_bCH₃ major, -OCH_aH_bCH₃ minor), 3.77-3.95 (m, 2H, -OCH_aH_bCH₃ minor, -OCH_aH_bCH₃ major), 4.07 (dd, J_2-3 = 10.2 Hz, J_3-4 = 7.7 Hz, 1 H, H-3) (minor), 4.34 (d, J4-₅ = 9.1 Hz, 1 H, H-5) (minor), 4.39-4.44 (m, 2H, H-4 minor, H-5 major), 4.50 (dd, J_3-4 = 9.5 Hz, J_4-5 = 6.2 Hz, 1 H, H-4) (major), 4.76 (d, Ji_-2 = 2.8 Hz, 1 H, H-1 ) (major), 4.79 (d, J_1-2 = 2.9 Hz, 1 H, H-1 ) (minor), 4.89 (d, J = 1 ,1 Hz, 1 H, H-7) (minor), 4.93 (s I, 1 H, H-7) (major).

¹³C NMR (CDCI₃, 100.6 MHz): δ 15.1 (-OCH₂CH₃) (minor), 15.2 (-OCH₂CH₃) (major), 56.7 (-OCH₃) (minor), 57.2 (-OCH₃) (major), 59.3 (-OCH₃) (mixture), 59.8 (-OCH₃) (major), 60.6 (-OCH₃) (minor), 65.0 (-OCH₂CH₃) (minor), 65.5 (-OCH₂CH₃) (major), 70.2 (C-5) (major), 72.4 (C-5) (minor), 75.9 (C-4) (major), 79.2 (C-4) (minor), 79.4 (C-3) (major), 79.8 (C-2 major, C-3 minor), 80.2 (C-2) (minor), 96.1 (C-7) (major), 97.2 (C-7) (minor), 98.7 (C-1 ) (major), 99.0 (C-1 ) (minor), 205.3 (C-6) (minor), 205.6 (C-6) (major).

IR (film) v (cm^“1): 1783 (C=0).

ESI-MS (pos. mode): m/z = 299 [M+Na]⁺, 331 [M+Na+MeOH]⁺.

Preparation of Methyl methyl-2,3-di-0-methyl-a-L-idopyranosiduronate (IXa)

; CHCI3)

To a solution of compound Villa (50 mg, 0.18 mmol, 1 .0 eq.) in dichloromethane (3 mL), under an argon atmosphere and cooled to 0°C, were added m-CPBA (77%, 120 mg, 0.54 mmol, 3.0 eq.) and NaHC0₃ (20 mg, 0.23 mmol, 1 .3 eq.). After 3 hours stirring the solvent was removed under vacuum. The resulting residue was dissolved in EtOAc (30 mL), extracted with distilled water (2 x 10 mL), and the aqueous phase was concentrated. The crude mixture was dissoved in methanol (10 mL), para-toluenesulfonic acid monohydrate was added (4 mg, 0.02 mmol, 0.1 eq.) then the reaction mixture was heated to reflux and the reaction monitored by ¹H NMR in deuterated methanol. After 8 hours the solvent was evaporated. The residue obtained was dissolved in DMF (5 mL) then triethylamine (28 μί, 0.20 mmol, 1 .1 eq.) and methyl iodide (56 μί, 0.90 mmol, 5 eq.) were added. After 3h30 the reaction mixture was concentrated, dissolved in EtOAc (30 mL) and the organic phase was washed with a 5% NaHC0₃ aqueous solution (2 x 10 mL), a 5% citric acid aqueous solution (2 x 10 mL) and brine (1 x 10 mL). The aqueous phase was extracted with dichloromethane (5 x 10 mL) and the combined organics were dried (MgS0₄), filtered and concentrated. Column chromatography (dichloromethane/ethyl acetate 85:15) afforded compound Xla as a colourless oil (25 mg, 0.10 mmol, 56%). ¹H NMR (CDCI3, 400 MHz): δ 3.41 (d I, J_2-3 = 3.5 Hz, 1 H, H-2), 3.47 (s, 3H, -OCH3), 3.56 (s, 3H, -OCH3), 3.57 (s, 3H, -OCH3), 3.69 (t, J_2-3 = J3-4 = 3.5 Hz, 1 H, H-3), 3.75-3.78 (m, 1 H, OH), 3.80 (s, 3H, -OCH₃), 3.97 (m, 1 H, H-4), 4.42 (d, J_4-5 = 1 .6 Hz, 1 H, H-5), 4.61 (d,

¹³C NMR (CDCI₃, 100.6 MHz): δ 52.4, 57.5, 58.4, 60.8 (4x-OCH₃), 67.7 (C-4), 74.8 (C-5), 77.2 (C-2), 77.5 (C-3), 100.9 (C-1 ), 169.6 (C=0).

Elemental analysis: Calculated: C: 48.00 ; H: 7.25. Found: C: 47.62 ; H: 7.15.

ESI-MS (pos. mode): m/z = 272 [M+Na]⁺.

IR (film) v (cm^“1): 3491 (O-H), 1765 (C=0). Example 2 :

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(2′,3′-di-0-methyl-p-D-glucopyranosyl- uronate)-a-D-glucopyranoside (IVb)

To a solution of the co

Me

(3.279 g, 5.00 mmol, 1.0 eq.) in a water/acetonitrile mixture (1 :1 , 300 mL) were added NaBr (105 mg, 1 .02 mmol, 0.2 eq.) and TEMPO (33 mg, 0.21 mmol, 0.04 eq.). The reaction mixture was cooled with the aid of an ice bath then a solution of NaOCI (13% v/v, 1 1.5 mL, 20.08 mmol, 4.0 eq.) was added. After 3 hours stirring at 0°C, NaOCI was added anew (13% v/v, 1 1 .5 mL, 20.08 mmol, 4.0 eq.). After two more hours ethanol was added (96% v/v, 20 mL), then the pH was reduced to 2-3 by addition of HCI (10% v/v). The solvent was evaporated and the residue obtained was suspended in DMF (40 mL) then triethylamine (2.8 mL, 2.032 g, 20.0 mmol, 4.0 eq.) and methyl iodide (6.2 mL, 14.136 g, 99.6 mmol, 20.0 eq.) were added. After 4 hours stirring at room temperature the solvent was evaporated and the residue obtained was dissolved in EtOAc (200 mL). The organic layer was washed with a 5% citric acid aqueous solution (1 χ 20 mL) and brine (1 χ 20 mL). The aqueous layer was extracted with dichloromethane (2 χ 20 mL). The combined organics were dried (MgS0₄), filtered and evaporated. The residue obtained was dissolved in DMF (20 mL), then triethylamine (1.4 mL, 1 .016 g, 10.0 mmol, 2.0 eq.) and methyl iodide (3.1 mL, 7.068 g, 49.8 mmol, 10.0 eq) were added. After 60 hours stirring at room temperature the solvent was evaporated and the residue obtained was dissolved in EtOAc (200 mL). The organic layer was washed with a 5% citric acid aqueous solution (2 x 20 mL) and brine (1 χ 20 mL). The organic layer was dried (MgS0₄), filtered and evaporated. Column chromatography (gradient hexane/ethyl acetate 80:20 – 50:50) gave compound IVb as a colourless oil which was dissolved in a diethyl ether/hexane mixture and evaporated at room temperature to afford a white solid (2.500 g, 3.66 mmol, 73%).

¹H NMR (CDCI₃, 400 MHz): δ 2.92 (d, 1 H), 2.93-2.98 (m, 2H), 3.41 (s, 3H), 3.48-3.76 (m, 5H), 3.51 (s, 3H), 3.60 (s, 3H), 3.63 (s, 3H), 3.87-3.98 (m, 3H), 4.36-4.41 (m, 1 H), 4.49- 5.08 (m, 6H), 4.61 (d, 1 H), 7.24-7.42 (m, 15H).

Elemental analysis: Calculated: C: 65.09 ; H: 6.79. Found: C: 65.29 ; H: 6.96.

ESI-MS (pos. mode): m/z = 705 [M+Na]⁺.

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(4′-0-(1 “-ethoxy-2″-propyn-1 “-yl)-2′,3′- di-0-methyl-p-D-glucopyranosyluronate)-a-D-glucopyranoside (Vb)

E F To a solution of compound IVb (385 mg, 0.56 mmol, 1 .0 eq.) in chloroform (stabilised with amylene, 30 mL) were added, under an argon atmosphere, P₂0₅ (410 mg, 2.80 mmol, 5.0 eq.) and propargylaldehyde diethylacetal (0.4 mL, 2.79 mmol, 5.0 eq.), then the reaction mixture was heated at reflux. After 5 hours stirring, the reaction mixture was filtered through a pad of Celite^® then the solvent was removed under vacuum. The crude mixture was suspended in EtOAc (60 mL), washed with a 5% NaHC0₃ aqueous solution (1 x 15 mL) and brine (1 x 15 mL). The organic layer was dried (MgS0₄), filtered, and evaporated. Column chromatography (gradient hexane/ethyl acetate 90:10 – 70:30) afforded compound Vb as a colourless oil (275 mg, 0.36 mmol, 64%) in a diastereomeric mixture (64:36).

¹H NMR (CDCI₃, 250 MHz): δ 1.17-1 .27 (m, 3H), 2.55 (d, 0.36H), 2.57 (d, 0.64H), 2.92- 3.08 (m, 2H), 3.38 (s, 3H), 3.49 (s, 1.92H), 3.50 (s, 1.08H), 3.57 (s, 1.08H), 3.59 (s, 1.92H), 3.60 (s, 1.92H), 3.62 (s, 1.08H), 3.44-3.97 (m, 10H), 4.35 (t, 1 H), 4.46-4.76 (m, 6H), 5.03 (d, 1 H), 5.32 (d, 0.36H), 5.56 (d, 0.64H), 7.21-7.42 (m, 15H).

Elemental analysis: Calculated: C: 65.95 ; H: 6.85. Found: C: 65.92 ; H: 6.75.

ESI-MS (pos. mode): m/z = 787 [M+Na]⁺.

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(4′-0-(1 “-ethoxy-2″-propyn-1 ” 1 ‘,2′,3′-tri-0-methyl-a-D-glucopyranosiduronic acid)-a-D-glucopyranoside (Vlb)

E F

To a solution of compound Vb (1.02 g, 1.33 mmol, 1.0 eq.) in EtOH/H₂0 (1 :1 , 100 mL) was added sodium hydroxide (82 mg, 2.05 mmol, 1.5 eq.). After 3 hours stirring at room temperature sodium hydroxide was added anew (27 mg, 0.68 mmol, 0.5 eq.). After an additional hour stirring the solvent was evaporated. The residue obtained was dissolved in water (40 mL). The pH of the aqueous layer was reduced to 2-3 with a 10% HCI aqueous solution then the layer was saturated with sodium chloride before extraction with dichloromethane (3 x 20 mL). The combined organics were dried (MgS0₄), filtered and removed under vacuum. Compound VIb was obtained without further purification as a white solid (930 mg, 1.24 mmol, 93%), in a diastereomeric mixture (63:37).

¹H NMR (CDCIs, 400 MHz): δ 1.17-1.28 (m, 3H), 2.65 (d, 0.63H), 2.68 (d, 0.37H), 2.90- 3.09 (m, 2H), 3.37 (s, 3H), 3.46 (s, 1.1 1 H), 3.57 (s, 1.89H), 3.58 (s, 1.1 1 H), 3.60 (s, 1.89H), 3.42-3.90 (m, 10H), 4.29 (d, 1 H), 4.46-4.97 (m, 7H), 5.44 (d, 0.37H), 5.61 (d, 0.63H), 7.28-7.44 (m, 15H).

ESI-MS (pos. mode): m/z = 773 [M+Na]⁺ , 795 [M-H+2Na]⁺ . ESI-MS (neg. mode): m/z = 749 [M-H]\

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(4′,7′-anhydro-6′-deoxy-6′-methylene-7′- ethoxy-2 3′-di-0-methyl-α-L-/^‘ o-heptopyranosyl)-α-D-glucopyranoside (Vllb)

To a solution of compound VIb (647 mg, 0.86 mmol, 1.0 eq.) in anhydrous THF (20 mL) and cooled to 0°C, were added IBCF (0.1 1 mL, 0.85 mmol, 1.0 eq.) and N- methylmorpholine (0.10 mL, 0.91 mmol, 1.1 eq.). After 10 minutes stirring, the flask was covered with aluminium foil, 2-mercaptopyridine /V-oxide sodium salt (512 mg, 3.43 mmol, 4.0 eq.) was added and the reaction mixture was stirred at ambient temperature. After 20 minutes anhydrous THF (100 mL) then ie f-butylthiol (0.18 mL, 1 .68 mmol, 2.0 eq.) were added. The aluminium foil was removed and the reaction mixture was irradiated and heated 15 minutes with a UV lamp (300W). The thiol excess was neutralized with a NaOCI aqueous solution (13%, 10 mL). The reaction mixture was concentrated then dissolved in EtOAc (100 mL), washed successively with a 5% NaHC0₃ aqueous solution (1 x 15 mL), a 5 % citric acid aqueous solution (1 x 15 mL) and brine (1 x 15 mL), then the aqueous layer was extracted with dichloromethane (2 x 20 mL). The combined organics were dried (MgS0₄), filtered and concentrated. Column chromatography (gradient hexane/ethyl acetate 90 :10 – 70:30) afforded compound Vllb as a colourless oil (251 mg, 0.36 mmol, 42%) in a mixture of diastereomers (61 :39).

¹H NMR (CDCI₃, 250 MHz): δ 1.18-1.29 (m, 3H), 2.90-3.07 (m, 1 H), 3.37 (s, 1.17H), 3.38 (s, 1 .83H), 3.46 (s, 3H), 3.54 (s, 1.83H), 3.60 (s, 1 .17H), 3.29-4.00 (m, 10H), 4.1 1 -4.26 (m, 1 H), 4.50-4.96 (m, 8H), 5.09-5.48 (m, 3H), 7.23-7.39 (m, 15H).

ESI-MS (pos. mode): m/z = 720 [M+Na]⁺ _.

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(4′,7′-anhydro-7′-ethoxy-2′,3′-di-0- methyl-a-L-/^‘ o-heptopyranosid-6′-ulosyl)-a-D-glucopyranoside (Vlllb)

Through a solution of compound Vllb (145 mg, 0.21 mmol, 1 .0 eq.) in anhydrous dichloromethane (10 mL), under an argon atmosphere and cooled to -78°C, was bubbled ozone (0.2 L/min, 1 10 V). When the solution had turned dark blue, oxygen was bubbled through in order to remove the excess ozone. When the solution became colorless dimethylsulfide (4 drops) was added and the solution was brought to room temperature. After 30 min stirring the reaction mixture was concentrated. Column chromatography (gradient hexane/ethyl acetate 90:10 – 60:40) afforded compound Vlllb as a white solid (100 mg, <67%) in a mixture of diastereomers (67:33).

¹H NMR (CDCI₃, 400 MHz): δ 1.21 -1 .28 (m, 3H), 2.93-3.07 (m, 1 H), 3.31-4.26 (m, 20H), 4.52-5.02 (m, 9H), 7.20-7.45 (m, 15H).

ESI-MS (pos. mode): m/z = 731 [M+Na]⁺ _.

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(methyl 2′,3′-di-0-methyl-a-L- idopyranosiduronate)-a-D-glucopyranoside (IXb)

To a solution of compound Vlllb (62 mg, 87 μηηοΙ, 1 .0 eq.) in dichloromethane (5 mL), under an argon atmosphere and cooled to 0°C, were added m-CPBA (77%, 58 mg, 259 μηηοΙ, 3.0 eq.) and NaHC0₃ (1 1 mg, 130 μηηοΙ, 1 .5 eq.). After 5 hours stirring the solvent was removed under vacuum. The reaction mixture was then dissolved in EtOAc (50 mL) and washed successively with a 5% NaHC0₃ aqueous solution (1 x 10 mL), a 5 % citric acid aqueous solution (1 x 10 mL) and brine (1 x 10 mL). The organic layer was dried (MgS0₄), filtered and concentrated. The crude mixture was dissolved in anhydrous methanol (10 mL) and sodium methoxide was added to reach pH = 10. After 30 minutes stirring at room temperature the reaction mixture was neutralized with Dowex^®, filtered through a pad of Celite^®, and concentrated. The residue obtained was dissolved in DMF (10 mL) then triethylamine (13 μί, 93 μηηοΙ, 1.1 eq.) and methyl iodide (27 μί, 434 μηηοΙ, 5.0 eq.) were added. After 2h30 stirring the reaction mixture was concentrated, dissolved in EtOAc (40 mL) and washed with a 5% citric acid aqueous solution (2 x 10 mL), a 5% NaHC0₃ aqueous solution (2 x 10 mL), and brine (1 x 10 mL). The organic layer was dried (MgS0₄), filtered and concentrated. Column chromatography (gradient hexane/ethyl acetate 60:40-50:50) afforded compound IXb as a colourless oil (12 mg, 18 μηηοΙ, 20% over two steps).

¹H NMR (CDCIs, 400 MHz): δ 3.23 (s, 3H), 3.20-3.25 (m, 1 H), 3.36 (s, 3H), 3.43 (s, 3H), 3.46 (s, 3H), 3.33-3.58 (m, 3H), 3.60-3.69 (m, 2H), 3.72-3.95 (m, 4H), 4.53-4.60 (m, 4H), 4.68-4.97 (m, 4H), 5.14 (s, 1 H), 7.24-7.37 (15H).

ESI-MS (pos. mode): m/z = 705 [M+Na]⁺ _.

……………

Volume 69, Issue 15, 15 April 2013, Pages 3149–3158

http://www.sciencedirect.com/science/article/pii/S0040402013003025

References

HEPARIN SODIUM

LAUNCHED 1937

9041-08-1 NA SALT
9005-49-6 (heparin)

Thromboliquine, Calciparine, Certoparin, Dalteparin, Fraxiparin, Heparinate, Multiparin, Novoheparin, Parnaparin

Unfractionated heparin (UH) is a heterogenous preparation of anionic, sulfated glycosaminoglycan polymers with weights ranging from 3000 to 30,000 Da. It is a naturally occurring anticoagulant released from mast cells. It binds reversibly to antithrombin III (ATIII) and greatly accelerates the rate at which ATIII inactivates coagulation enzymes thrombin (factor IIa) and factor Xa. UH is different from low molecular weight heparin (LMWH) in the following ways: the average molecular weight of LMWH is about 4.5 kDa whereas it is 15 kDa for UH; UH requires continuous infusions; activated partial prothrombin time (aPTT) monitoring is required when using UH; and UH has a higher risk of bleeding and higher risk of osteoporosis in long term use. Unfractionated heparin is more specific than LMWH for thrombin. Furthermore, the effects of UH can typically be reversed by using protamine sulfate.

Unfractionated heparin is indicated for prophylaxis and treatment of venous thrombosis and its extension, prevention of post-operative deep venous thrombosis and pulmonary embolism and prevention of clotting in arterial and cardiac surgery. In cardiology, it is used to prevent embolisms in patients with atrial fibrillation and as an adjunct antithrombin therapy in patients with unstable angina and/or non-Q wave myocardial infarctions (i.e. non-ST elevated acute coronary artery syndrome) who are on platelet glycoprotein (IIb/IIIa) receptor inhibitors. Additionally, it is used to prevent clotting during dialysis and surgical procedures, maintain the patency of intravenous injection devices and prevent in vitro coagulation of blood transfusions and in blood samples drawn for laboratory values.

Indication: For anticoagulant therapy in prophylaxis and treatment of venous thrombosis and its extension, for prevention of post-operative deep venous thrombosis and pulmonary embolism and for the prevention of clotting in arterial and cardiac surgery.
Mechanism of action: The mechanism of action of heparin is antithrombin-dependent. It acts mainly by accelerating the rate of the neutralization of certain activated coagulation factors by antithrombin, but other mechanisms may also be involved. The antithrombotic effect of heparin is well correlated to the inhibition of factor Xa. Heparin interacts with antithrombin III, prothrombin and factor X.

Heparin (from Ancient Greek ηπαρ (hepar), liver), also known as unfractionated heparin, a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule.^[3] It can also be used to form an inner anticoagulant surface on various experimental and medical devices such as test tubes and renal dialysis machines.

Although it is used principally in medicine for anticoagulation, its true physiological role in the body remains unclear, because blood anticoagulation is achieved mostly by heparan sulfate proteoglycans derived from endothelial cells.^[4] Heparin is usually stored within the secretory granules of mast cells and released only into the vasculature at sites of tissue injury. It has been proposed that, rather than anticoagulation, the main purpose of heparin is defense at such sites against invading bacteria and other foreign materials.^[5] In addition, it is conserved across a number of widely different species, including some invertebrates that do not have a similar blood coagulation system.

HEPARIN

Heparin structure

Native heparin is a polymer with a molecular weight ranging from 3 to 30 kDa, although the average molecular weight of most commercial heparin preparations is in the range of 12 to 15 kDa.^[6] Heparin is a member of the glycosaminoglycan family of carbohydrates (which includes the closely related molecule heparan sulfate) and consists of a variably sulfated repeating disaccharide unit.^[7] The main disaccharide units that occur in heparin are shown below. The most common disaccharide unit is composed of a 2-O-sulfated iduronic acid and 6-O-sulfated, N-sulfated glucosamine, IdoA(2S)-GlcNS(6S). For example, this makes up 85% of heparins from beef lung and about 75% of those from porcine intestinal mucosa.^[8] Not shown below are the rare disaccharides containing a 3-O-sulfated glucosamine (GlcNS(3S,6S)) or a free amine group (GlcNH₃⁺). Under physiological conditions, the ester and amide sulfate groups are deprotonated and attract positively charged counterions to form a heparin salt. Heparin is usually administered in this form as an anticoagulant.

One unit of heparin (the “Howell unit”) is an amount approximately equivalent to 0.002 mg of pure heparin, which is the quantity required to keep 1 ml of cat’s blood fluid for 24 hours at 0°C.^[9]

Abbreviations

• GlcA = β-D-glucuronic acid
• IdoA = α-L-iduronic acid
• IdoA(2S) = 2-O-sulfo-α-L-iduronic acid
• GlcNAc = 2-deoxy-2-acetamido-α-D-glucopyranosyl
• GlcNS = 2-deoxy-2-sulfamido-α-D-glucopyranosyl
• GlcNS(6S) = 2-deoxy-2-sulfamido-α-D-glucopyranosyl-6-O-sulfate

Three-dimensional structure

The three-dimensional structure of heparin is complicated because iduronic acid may be present in either of two low-energy conformations when internally positioned within an oligosaccharide. The conformational equilibrium is influenced by sulfation state of adjacent glucosamine sugars.^[10] Nevertheless, the solution structure of a heparin dodecasaccharide composed solely of six GlcNS(6S)-IdoA(2S) repeat units has been determined using a combination of NMR spectroscopy and molecular modeling techniques.^[11] Two models were constructed, one in which all IdoA(2S) were in the ²S₀ conformation (A and B below), and one in which they are in the ¹C₄ conformation (C and D below). However, no evidence suggests that changes between these conformations occur in a concerted fashion. These models correspond to the protein data bank code 1HPN.

In the image above:

• A = 1HPN (all IdoA(2S) residues in ²S₀ conformation) Jmol viewer
• B = van der Waals radius space filling model of A
• C = 1HPN (all IdoA(2S) residues in ¹C₄ conformation) Jmol viewer
• D = van der Waals radius space filling model of C

In these models, heparin adopts a helical conformation, the rotation of which places clusters of sulfate groups at regular intervals of about 17 angstroms (1.7 nm) on either side of the helical axis.

Medical use

A sample of Heparin Sodium for injection

Heparin is a naturally occurring anticoagulant produced by basophils and mast cells.^[12] Heparin acts as an anticoagulant, preventing the formation of clots and extension of existing clots within the blood. While heparin does not break down clots that have already formed (unlike tissue plasminogen activator), it allows the body’s natural clot lysis mechanisms to work normally to break down clots that have formed. Heparin is generally used for anticoagulation for the following conditions:

• Acute coronary syndrome, e.g., NSTEMI
• Atrial fibrillation
• Deep-vein thrombosis and pulmonary embolism
• Cardiopulmonary bypass for heart surgery
• ECMO circuit for extracorporeal life support
• Hemofiltration
• Indwelling central or peripheral venous catheters

Mechanism of action

Heparin and its low-molecular-weight derivatives (e.g., enoxaparin, dalteparin, tinzaparin) are effective at preventing deep vein thromboses and pulmonary emboli in patients at risk,^[13][14] but no evidence indicates any one is more effective than the other in preventing mortality.^[15] Heparin binds to the enzyme inhibitor antithrombin III (AT), causing a conformational change that results in its activation through an increase in the flexibility of its reactive site loop.^[16] The activated AT then inactivates thrombin and other proteases involved in blood clotting, most notably factor Xa. The rate of inactivation of these proteases by AT can increase by up to 1000-fold due to the binding of heparin.^[17]

AT binds to a specific pentasaccharide sulfation sequence contained within the heparin polymer:

GlcNAc/NS(6S)-GlcA-GlcNS(3S,6S)-IdoA(2S)-GlcNS(6S)

The conformational change in AT on heparin-binding mediates its inhibition of factor Xa. For thrombin inhibition, however, thrombin must also bind to the heparin polymer at a site proximal to the pentasaccharide. The highly negative charge density of heparin contributes to its very strong electrostatic interaction with thrombin.^[3] The formation of a ternary complex between AT, thrombin, and heparin results in the inactivation of thrombin. For this reason, heparin’s activity against thrombin is size-dependent, with the ternary complex requiring at least 18 saccharide units for efficient formation.^[18] In contrast, antifactor Xa activity requires only the pentasaccharide binding site.

Chemical structure of fondaparinux

This size difference has led to the development of low-molecular-weight heparins (LMWHs) and, more recently, to fondaparinux as pharmaceutical anticoagulants. LMWHs and fondaparinux target antifactor Xa activity rather than antithrombin activity, with the aim of facilitating a more subtle regulation of coagulation and an improved therapeutic index. The chemical structure of fondaparinux is shown above. It is a synthetic pentasaccharide, whose chemical structure is almost identical to the AT binding pentasaccharide sequence that can be found within polymeric heparin and heparan sulfate.

With LMWH and fondaparinux, the risk of osteoporosis and heparin-induced thrombocytopenia (HIT) is reduced. Monitoring of the activated partial thromboplastin time is also not required and does not reflect the anticoagulant effect, as APTT is insensitive to alterations in factor Xa.

Danaparoid, a mixture of heparan sulfate, dermatan sulfate, and chondroitin sulfate can be used as an anticoagulant in patients having developed HIT. Because danaparoid does not contain heparin or heparin fragments, cross-reactivity of danaparoid with heparin-induced antibodies is reported as less than 10%.^[19]

The effects of heparin are measured in the lab by the partial thromboplastin time (aPTT), one of the measures of the time it takes the blood plasma to clot. Partial thromboplastin time should not be confused with prothrombin time, or PT, which measures blood clotting time through a different pathway of the coagulation cascade.

Administration

Heparin is given parenterally because it is not absorbed from the gut, due to its high negative charge and large size. It can be injected intravenously or subcutaneously (under the skin); intramuscular injections (into muscle) are avoided because of the potential for forming hematomas. Because of its short biologic half-life of about one hour, heparin must be given frequently or as a continuous infusion. Unfractionated heparin has a half-life of about one to two hours after infusion, ^[20] whereas LMWH has a half-life of four to five hours.^[21] The use of LMWH has allowed once-daily dosing, thus not requiring a continuous infusion of the drug. If long-term anticoagulation is required, heparin is often used only to commence anticoagulation therapy until an oral anticoagulant e.g. warfarin takes effect.

Details of administration are available in clinical practice guidelines by the American College of Chest Physicians:^[22]

• Non-weight-based heparin dose adjustment
• Weight-based-heparin dose adjustment

Production

Pharmaceutical-grade heparin is derived from mucosal tissues of slaughtered meat animals such as porcine (pig) intestines or bovine (cattle) lungs.^[23] Advances to produce heparin synthetically have been made in 2003 and 2008.^[24]

Protamine sulfate (1 mg per 100 units of heparin that had been given over the past four hours) has been given to counteract the anticoagulant effect of heparin.^[26]

Heparin is one of the oldest drugs currently in widespread clinical use. Its discovery in 1916 predates the establishment of the Food and Drug Administration of the United States, although it did not enter clinical trials until 1935.^[27] It was originally isolated from canine liver cells, hence its name (hepar or “ήπαρ” is Greek for “liver”). Heparin’s discovery can be attributed to the research activities of Jay McLean and William Henry Howell.

In 1916, McLean, a second-year medical student at Johns Hopkins University, was working under the guidance of Howell investigating procoagulant preparations, when he isolated a fat-soluble phosphatide anticoagulant in canine liver tissue. In 1918, Howell coined the term ‘heparin’ for this type of fat-soluble anticoagulant. In the early 1920s, Howell isolated a water-solublepolysaccharide anticoagulant, which was also termed ‘heparin’, although it was distinct from the phosphatide preparations previously isolated. McLean’s work as a surgeon probably changed the focus of the Howell group to look for anticoagulants, which eventually led to the polysaccharide discovery.

In the 1930s, several researchers were investigating heparin. Erik Jorpes at Karolinska Institutet published his research on the structure of heparin in 1935,^[28] which made it possible for the Swedish company Vitrum AB to launch the first heparin product for intravenous use in 1936. Between 1933 and 1936, Connaught Medical Research Laboratories, then a part of the University of Toronto, perfected a technique for producing safe, nontoxic heparin that could be administered to patients in a salt solution. The first human trials of heparin began in May 1935, and, by 1937, it was clear that Connaught’s heparin was a safe, easily available, and effective blood anticoagulant. Prior to 1933, heparin was available, but in small amounts, and was extremely expensive, toxic, and, as a consequence, of no medical value.^[29]

A posthumous attempt to nominate McLean for a Nobel Prize failed

Heparin Sodium Injection, USP is a sterile, nonpyrogenic solution of heparin sodium (derived from porcine intestinal mucosa) in water for injection. Each container contains 10000, 12500, 20000 or 25,000 USP Heparin Units; 40 or 80 mg sodium chloride added to render isotonic (see HOW SUPPLIEDsection for various sizes and strength). May contain sodium hydroxide and/or hydrochloric acid for pH adjustment. pH 6.0 (5.0 to 7.5).

The solution contains no bacteriostat, antimicrobial agent or added buffer and is intended for use only as a single-dose injection. When smaller doses are required, the unused portion should be discarded.

Heparin sodium in the ADD-Vantage™ system is intended for intravenous administration only after dilution.

Heparin Sodium, USP is a heterogenous group of straight-chain anionic mucopolysaccharides, called glycosamino-glycans having anticoagulantproperties. Although others may be present, the main sugars occurring in heparin are: (1) α- L-iduronic acid 2-sulfate, (2) 2-deoxy-2-sulfamino-α-D-glucose-6-sulfate, (3) β-D-glucuronic acid, (4) 2-acetamido-2-deoxy-α-D-glucose, and (5) α-L-iduronic acid. These sugars are present in decreasing amounts, usually in the order (2) > (1) > (4) > (3) > (5), and are joined by glycosidic linkages, forming polymers of varying sizes. Heparin is strongly acidic because of its content of covalently linked sulfate and carboxylic acid groups. In heparin sodium, the acidic protons of the sulfate units are partially replaced by sodium ions. The potency is determined by a biological assay using a USP reference standard based on units of heparin activity per milligram.

Structure of Heparin Sodium (representative subunits):

………………..

http://www.medgadget.com/2008/08/on_the_road_to_a_fully_synthetic_heparin.html

…………

The chemoenzymatic synthesis of heparin from E. coli’s carbohydrate coat

Now, Linhardt’s team – who were also the first to identify the contaminant in the tainted batches as oversulfated chondroitin sulfate – have come up with a potentially safer way to produce heparin. The researchers grew flasks of the gut bacteria E. coli, then converted its naturally produced carbohydrate coat to heparin in just a few steps using enzymes and chemical treatment.

Linhardt says the key to the procedure was starting with the carbohydrate capsule that E coli produces to hide itself from the human immune system. The capsule is made from heparosan – a polysachharide that is already quite similar to heparin.

The team first chemically removed acetyl groups from the heparosan with sodium hydroxide and added a sulfate group using sulfur trioxide trimethylamine. Then, using four enzymes found in all mammals that produce heparin, they introduced further modifications, including the addition of three more sulfates at different positions on the molecule to get to heparin.

The checked the structure of the compound using NMR and showed that the synthetic compound could stop blood clotting as well as heparin derived from animals. To date, however, the team have only made a total of around 100mg of pure heparin – barely enough for a single dose. That is still a million times more than produced by a 2003 total synthesis of heparin, from researchers at the Massachusetts Institute of Technology, US.

1. Heparin Sodium injection
2.  heparin. In: Lexi-Drugs Online [database on the Internet]. Hudson (OH): Lexi-Comp, Inc.; 2007 [cited 2/10/12]. Available from: http://online.lexi.com. subscription required to view.
3.  Cox, M.; Nelson D. (2004). Lehninger, Principles of Biochemistry (4). Freeman. p. 1100. ISBN 0-7167-4339-6.
4.  Marcum JA, McKenney JB. et al. (1986). “Anticoagulantly active heparin-like molecules from mast cell-deficient mice”.Am. J. Physiol. 250 (5 Pt 2): H879–888. PMID 3706560.
5.  Nader, HB et al.; Chavante, S.F.; Dos-Santos, E.A.; Oliveira, F.W.; De-Paiva, J.F.; Jerônimo, S.M.B.; Medeiros, G.F.; De-Abreu, L.R.D. et al. (1999). “Heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates?”. Braz. J. Med. Biol. Res. 32 (5): 529–538. doi:10.1590/S0100-879X1999000500005. PMID 10412563.
6.  Francis CW, Kaplan KL (2006). “Chapter 21. Principles of Antithrombotic Therapy”. In Lichtman MA, Beutler E, Kipps TJ, et al. Williams Hematology (7th ed.). ISBN 978-0-07-143591-8.
7.  Bentolila, A. et al.. “Synthesis and heparin-like biological activity of amino acid-based polymers” (Subscription required). Wiley InterScience. Retrieved 2008-03-10.
8.  Gatti, G., Casu, B. et al. (1979). “Studies on the Conformation of Heparin by ^lH and ¹³C NMR Spectroscopy” (PDF). Macromolecules 12 (5): 1001–1007. Bibcode:1979MaMol..12.1001G.doi:10.1021/ma60071a044.
   
9.  “Online Medical Dictionary”. Centre for Cancer Education. 2000. Retrieved 2008-07-11.
   
10.  Ferro D, Provasoli A, et al. (1990). “Conformer populations of L-iduronic acid residues in glycosaminoglycan sequences”. Carbohydr. Res. 195 (2): 157–167.doi:10.1016/0008-6215(90)84164-P. PMID 2331699.
11.  Mulloy B, Forster MJ, Jones C, Davies DB. (1 January 1993). “N.m.r. and molecular-modelling studies of the solution conformation of heparin”. Biochem. J. 293 (Pt 3): 849–858. PMC 1134446. PMID 8352752.
12.  Guyton, A. C.; Hall, J. E. (2006). Textbook of Medical Physiology (11). Elsevier Saunders. p. 464. ISBN 0-7216-0240-1.
13.  Agnelli G, Piovella F, Buoncristiani P et al. (1998). “Enoxaparin plus compression stockings compared with compression stockings alone in the prevention of venous thromboembolism after elective neurosurgery”. N Engl J Med 339 (2): 80–5. doi:10.1056/NEJM199807093390204.PMID 9654538.
14.  Bergqvist D, Agnelli G, Cohen AT et al. (2002). “Duration of prophylaxis against venous thromboembolism with enoxaparin after surgery for cancer”. N Engl J Med 346(13): 975–980. doi:10.1056/NEJMoa012385.PMID 11919306.
15.  Handoll HHG, Farrar MJ, McBirnie J, Tytherleigh-Strong G, Milne AA, Gillespie WJ (2002). “Heparin, low molecular weight heparin and physical methods for preventing deep vein thrombosis and pulmonary embolism following surgery for hip fractures”. In Handoll, Helen HG. Cochrane Database Syst Rev 4 (4): CD000305.doi:10.1002/14651858.CD000305. PMID 12519540.
16.  Chuang YJ, Swanson R. et al. (2001). “Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin”. J. Biol. Chem. 276 (18): 14961–14971. doi:10.1074/jbc.M011550200. PMID 11278930.
17. Bjork I, Lindahl U. (1982). “Mechanism of the anticoagulant action of heparin”. Mol. Cell. Biochem. 48 (3): 161–182.doi:10.1007/BF00421226. PMID 6757715.
18.  Petitou M, Herault JP, Bernat A, Driguez PA et al. (1999). “Synthesis of Thrombin inhibiting Heparin mimetics without side effects”. Nature 398 (6726): 417–422.Bibcode:1999Natur.398..417P. doi:10.1038/18877.PMID 10201371.
19.  Shalansky, Karen. DANAPAROID (Orgaran) for Heparin-Induced Thrombocytopenia. Vancouver Hospital & Health Sciences Centre, February 1998 Drug & Therapeutics Newsletter. Retrieved on 8 January 2007.
20.  Eikelboom JW, Hankey GJ (2002). “Low molecular weight heparins and heparinoids”. The Medical Journal of Australia 177 (7): 379–383. PMID 12358583.
21.  Weitz JI (2004). “New anticoagulants for treatment of venous thromboembolism”. Circulation 110 (9 Suppl 1): I19–26. doi:10.1161/01.CIR.0000140901.04538.ae.PMID 15339877.
22.  Hirsh J, Raschke R (2004). “Heparin and low-molecular-weight heparin: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy”. Chest 126 (3 Suppl): 188S–203S.doi:10.1378/chest.126.3_suppl.188S. PMID 15383472.
23.  Linhardt RJ, Gunay NS. (1999). “Production and Chemical Processing of Low Molecular Weight Heparins”. Sem. Thromb. Hem. 3: 5–16. PMID 10549711.
24.  Bhattacharya, Ananyo (August 2008). “Flask synthesis promises untainted heparin”. Chemistry World. Royal Society of Chemistry. Retrieved 6 February 2011.
25.  Kusmer, Ken (20 September 2006). “3rd Ind. preemie infant dies of overdose”. Fox News (Associated Press). Retrieved 2007-01-08.
26.  Internal medicine, Jay H. Stein, page 635
27.  Linhardt RJ. (1991). “Heparin: An important drug enters its seventh decade”. Chem. Indust. 2: 45–50.
28.  Jorpes E (August 1935). “The chemistry of heparin”. The Biochemical Journal 29 (8): 1817–30. PMC 1266692.PMID 16745848.
29.  Rutty, CJ. “Miracle Blood Lubricant: Connaught and the Story of Heparin, 1928–1937″. Health Heritage Research Services. Retrieved 2007-05-21.
    

Heparin, a highly sulfated glycosaminoglycan (GAG), is used extensively as an anticoagulant.  It consists of repeating disaccharide units, containing iduronic acid (or glucuronic acid) and glucosamine, exhibiting variable degrees of sulfation.  Heparin, and its analogues, are used during surgery and dialysis, and are often used to coat indwelling catheters and other devices where the vascular system is exposed.  Administered parenterally, often continuously due to its short half-life, over 0.5 billion doses are required per year.  Currently obtained from mucosal tissue of meat animals, mainly porcine intestine, and to a lesser extent bovine lung, its early stage production is poorly controlled, due to the source of the material (Figure 1).  This problem came into sharp focus in 2008 when the presence of contaminating over-sulfated chondroitin sulfate in heparin, sourced from pigs, resulted in almost 100 deaths in the USA.  This, coupled with the fact that only two doses are obtained per animal means that the demand for alternative and more controlled sources of heparin is high.

Figure.1. Heparin ProductionIn an attempt to mimic the natural processes, which occur in mammalian synthesis of heparan sulfate, a less sulfated form of heparin was first examined (Figures 1 & 2).

Figure 2. Biosynthetic pathway of HS: The biosynthetic pathway includes the biosynthesis of polysaccharide backbone as well as the modification steps. The synthesis is initiated with a tetrasaccharide linkage region that contains xylose-galactose-galactose-glucuronic acid. The backbone is synthesized by HS polymerase. The backbone polysaccharide is then modified via five enzymatic modification steps. The modification site at each step is highlighted in a blue box²Based on this pathway, a biosynthetic pathway was designed that began with bacterial synthesis of the backbone structure. Escherichia coli K5 synthesizes a polysaccharide capsule consisting of repeating units of [(→4) β-D-glucuronic acid (GlcA) (1→4) N-acetyl-α-D-glucosamine (GlcNAc) (1→)]_n,¹ known as heparosan (Figures 2 & 3). Since heparosan is a precursor of heparin and heparan sulfate in eukaryotes, this provides an ideal starting point of the bioengineered heparin production process.

Figure.3. (A) The structure of heparosan disaccharide unit. (B) the structures of the major and minor variable repeating disaccharides comprising heparin where X = SO^3- or H and Y = SO^3- or COCH₃.¹

The fermentation process involves the use of glucose as the sole carbon source and ammonium chloride as the sole nitrogen source.  Initially developed and optimized on a 500mL scale, the process was then scaled up to a 3L fermentation and is currently at the 15L scale.  Using a fed-batch fermentation, with exponential feeding, a growth rate of 0.12h^-1 was achieved with a yield of 15gL^-1 heparosan.

Figure.4. Time course of dry cell weight (g/L) and heparosan concentration in the fermentation supernatant (g/L) during the fermentation in a 20 L fermentor¹

Once extracted from the cell and purified the heparosan polysaccharide undergoes a series of chemoenzymatic modifications to make heparin.  The first step is performed chemically and all other steps in the process use the enzymes listed in Figure 2 (above), which have been cloned and overexpressed in E. coli.The heparosan polysaccharide generated through this process is analyzed by polyacrylamide gel electrophoresis (PAGE), 1D- and 2D-NMR and disaccharide analysis is done using LC-MS of heparinase-digested polysaccharide. At each point in the process the polysaccharide is analyzed by a combination of these methods.References1. Z.Wang, J.S.Dordick, & R.J.Linhardt, “Escherichia coli K5 heparosan fermentation and improvement by genetic engineering.“ Bioengineered Bugs 2, 1-5 (2011)2. R. Liu,  Y.Xu,  M.Chen,  M.Weiwer,  X.Zhou,  A.S.Bridges,  P.L.DeAngelis,  Q.Zhang,  R.J.Linhardt,  J.Liu,  ”Chempenzymatic  design of  heparan  sulfate  oligosaccharides” ,  Journal of  Biological  Chemistry,  285,  34240-342

3. Z.Wang, M.Ly, F.Zhang, W. Zhong, A.Suen, A.M.Hickey, J.S.Dordick, R.J.Linhardt,”E. coli K5 fermentation and the preparation of heparosan, a bioengineered heparin precursor“, Biotechnol. Bioeng. 107, 964-973 (2010).

1.  M.Ly, Z.Wang, T.N.Laremore, F.Zhang, W.Zhong, D.Pu, D.V.Zagorevski, J.S.Dordick, R.J.Linhardt, “Analysis of E. coli K5 capsular polysaccharide heparosan.” Analytical and Bioanalytical Chemistry 399, 737-745 (2011).

Idrabiotaparinux

•  抗血栓治療中の出血率を低下させるためのイドラビオタパリナックスの使用

(biotinylated idraparinux, SSR-126517, SSR-126517E)

Idrabiotaparinux has an attached biotin moiety at the non-reducing end unit, which allows its neutralisation with avidin, an egg-derived protein with low antigenicity. This compound is currently investigated in clinical trials for prevention of recurrent VTE in patients with acute pulmonary embolism. The future of idrabiotaparinux depends also on the safety and efficacy of avidin.
Symptomatic deep vein thrombosis (DVT) and/or pulmonary embolism (PE) – treatment and secondary prevention of recurrent venous thromboembolism (VTE).

Idrabiotaparinux (biotinylated idraparinux, SSR-126517, SSR-126517E) is a long-acting selective pentasaccharide indirect factor Xa coagulation inhibitor, administered by once weekly subcutaneous (SC) injection at a dose of 3mg in patients without severe renal insufficiency and, after an initial dose of 3mg, at 1.8mg in those with renal insufficiency.

Warfarin, heparin and their derivatives have been the traditional anticoagulants used for prophylaxis and treatment of venous thromboembolism. While the modern clinician is familiar with the efficacy and pharmacokinetics of these agents, their adverse effects have provided the impetus for the development of newer anticoagulants with improved safety, ease of administration, more predictable pharmacodynamics and comparable efficacy. Research into haemostasis and the coagulation cascade has made the development of these newer anticoagulants possible.

These drugs include the factor Xa inhibitors and IIa (thrombin) inhibitors. Direct and indirect factor Xa inhibitors are being developed with a relative rapid onset of action and stable pharmacokinetic profiles negating the need for close monitoring; this potentially makes them a more attractive option than heparin or warfarin. Examples of direct factor Xa inhibitors include apixaban, rivaroxaban, otamixaban, betrixaban and edoxaban. Examples of indirect factor Xa inhibitors include fondaparinux, idraparinux and idrabiotaparinux.

Direct thrombin inhibitors (factor IIa inhibitors) were developed with the limitations of standard heparin and warfarin in mind. Examples include recombinant hirudin (lepirudin), bivalirudin, ximelagatran, argatroban, and dabigatran etexilate. This review will discuss emerging novel anticoagulants and their use for the prophylaxis and management of venous thromboembolism, for stroke prevention in nonvalvular atrial fibrillation and for coronary artery disease.

Idrabiotaparinux is intended as a substitute for current long-term oral anticoagulation (e.g. with warfarin) and has no known food or drug interactions, no need for overlapping with other anticoagulants or for laboratory blood monitoring.
Idrabiotaparinux has superseded the development and marketing of the non-biotinylated idraparinux. Idrabiotaparinux is also in phase III clinical trials for the prevention of stroke in patients with atrial fibrillation (AF).
Idrabiotaparinux will be the first once a week anticoagulant for the treatment of patients with VTE. It is intended to provide a predictable response with fixed dosing, no interactions with food, no requirement for overlapping with other therapy and no routine laboratory monitoring.
Developer  Sanofi-aventis.

Standard treatment of venous thromboembolism,including deep vein thrombosis and pulmonary
embolism, is started with a rapidly acting parenteral anticoagulant such as heparin or low-molecular-weight
heparin for at least 5 days and is overlapped with a Vitamin K antagonist such as warfarin.

Warfarin is then continued for at least 3 months. Although eff ective, this drug has important limitations. Lifestyle changes are necessary because of interactions with food, alcohol,and other drugs, and the unpredictable anticoagulant eff ect of warfarin necessitates frequent coagulation monitoring and dose adjustments to optimise the balance between effi cacy and safety. Warfarin reduces the risk of recurrent venous thromboembolism by up to 90%, but there is a catch-up eff ect if warfarin is stopped in patients with unprovoked venous thromboembolism. This eff ect means that, by 2 years,the risk of recurrence in patients treated for 3 months is akin to that in patients treated for 12 months.

Consequently, some experts recommend life-long warfarin therapy for patients with unprovoked venousthromboembolism. The complexity of such treatment has prompted the development of new oral and parenteral anticoagulants that are more convenient to administer than is warfarin Idraparinux is a synthetic pentasaccharide that accel erates antithrombin-dependent inhibition of factor Xa and has a half-life of about 80 h. When compared with conventional anticoagulation therapy,

idraparinux given once-weekly by subcutaneous injection was non-inferior for treatment of deep vein thrombosis,but was inferior for treatment of pulmonary embolism. In patients with venous thromboembolism who received a 6 month course of anticoagulant treatment, idraparinux was better than was placebo for prevention of recurrent venous thromboembolism.6However, when compared with warfarin for stroke prevention in patients with atrial fi brillation, there was an excess of major bleeding with idraparinux (including intracranial haemorrhage).7Prompted by these safety concerns, idrabiotaparinux was developed as a replacement for idraparinux.

Idrabiotaparinux (International Non-proprietary Name), or SSR126517 (laboratory code), is developed by sanofi-aventis as the first long-acting anticoagulant administered once-weekly by subcutaneous route, with the unique property to be almost instantly and specifically neutralizable by intravenous administration of avidin. It is developed as an alternative to vitamin K antagonists (VKA). Idrabiotaparinux is the biotinylated pentasaccharide corresponding to the structure depicted below.

The pentasaccharide structure of idrabiotaparinux is the same as idraparinux, another antithrombotic agent developed by sanofi-aventis (see structure below). However in idrabiotaparinux, the presence of a biotin hook covalently linked to the first saccharidic unit enables the compound to be neutralized by avidin or streptavidin, as described in the international patent application WO 02/24754.

Idraparinux

In the EQUINOX trial, which enrolled 757 patients with DVT treated for 6 months with equimolar doses of either idrabiotaparinux or idraparinux, the administration of idrabiotaparinux was demonstrated to provide bioequipotent results to idraparinux in terms of pharmacokinetics and pharmacodynamics, in patients with symptomatic deep venous thrombosis (Journal of Thrombosis and Haemostasis, 2010, Vol. 9, p. 92-99). The results of this bioequipotency trial indicated that idrabiotaparinux could be a suitable treatment for patients with deep venous thrombosis. However, the apparent failure of idraparinux in patients with pulmonary embolism indicated the need for a formal evaluation of idrabiotaparinux in this patient group (N. Eng. J. Med., 2007, Vol. 357, p. 1094-104).

IDRABIOTAPARINUX

It has now been demonstrated, in a phase III study involving 3202 patients with pulmonary embolism, that idrabiotaparinux is a safe and effective drug in the treatment of pulmonary embolism in patients with or without deep venous thrombosis and in the secondary prevention of venous thromboembolic events in said patients. The invention therefore relates to idrabiotaparinux for use in the treatment of pulmonary embolism in patients with or without deep venous thrombosis and the secondary prevention of venous thromboembolic events in said patients, wherein the efficacy and safety of said uses are clinically proven by a phase III clinical trial. According to the instant invention, the terms below have the following meanings:

“idrabiotaparinux” designates the sodium salt of this compound, as defined above, or any other pharmaceutically acceptable salt thereof;

-a “phase III clinical trial” refers to an international, multicenter, randomized, double-blind, double-dummy, parallel group study involving a large patients group (3202 patients in the instant invention), aiming at being the definitive assessment of how effective and safe the drug is, in comparison with current standard treatment; – “deep venous thrombosis” refers to a blood clot in a deep vein of the lower limbs;

new polysaccharides of the invention, are comparable to the oligosaccharides of the prior art antithrombotic activity. But they also have the advantage of being quickly neutralized by a specific antidote in an emergency. This specific antidote avidin (The Merck Index, Twelfth Edition, 1996, MN 920, pages 151-152) or streptavidin, two tetrameric protein with respective masses equal to approximately 66 000 and 60 000 Da, which have a very high affinity for biotin. In general, the invention relates to synthetic polysaccharides antithrombotic activity has at least one covalent bond with biotin or a biotin derivative. As a derivative of biotin include the biotin derivatives listed in the catalog Pierce 1999-2000 pages 62-81, for example 6-biotinamido hexanoate,

you,

or 2-biotinamido éthanethiole

Patent application WO 02/24754 describes synthetic polysaccharides which have a covalent bond with biotin (hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid) or with a biotin derivative. Such polysaccharides have an antithrombotic activity which means that they can be used as anticoagulants, and also have the advantage of being able to be rapidly neutralized with a specific antidote, in an emergency situation. This specific antidote is avidin (The Merck Index, Twelfth edition, 1996, M.N. 920, pages 151-152) or streptavidin, two tetrameric proteins of respective weights equal to approximately 66 000 and 60 000 Da, which have a very strong affinity for biotin.

Patent application WO 02/24754 describes in particular the following compound, known as idrabiotaparinux:

In the mammalian body, idrabiotaparinux is partly metabolized at the level of the amide bond adjacent to the biotin group, thus producing a pentasaccharide compound bearing an amine chain —NH—CO—(CH₂)₅—NH₂ on the first glucosamine unit, as described in patent application WO 2010/023374.

It may be desirable, in particular in the context of clinical developments of molecules of pharmaceutical interest, to limit or even prevent the metabolization of compounds of this type.

Novel polysaccharides with structures analogous to some of those described in patent application WO 02/24754 have now been identified, which polysaccharides have antithrombotic properties and a neutralization capacity, for example via avidin, which are comparable to those described in that patent application, but which also have improved metabolic stability.

Generally, the invention therefore relates to synthetic polysaccharides with antithrombotic activity having at least one covalent bond with biotin or a biotin derivative, characterized in that said covalent bond is resistant to metabolic cleavage and comprises a linkage X chosen from —O—, —N(R)—, —N(R)—CO— and —N(R′)—CO—N(R″)—, in which R is an alkyl group and R′ and R″, which may be identical or different, are, independently of one another, hydrogen atoms or alkyl groups.

For the purposes of the present invention, and unless otherwise mentioned in the text, the term “alkyl” is intended to mean a linear or branched, saturated aliphatic group comprising from 1 to 6 carbon atoms, and advantageously a methyl group.

Biotin, or hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid, is the compound having the following formula:

By way of biotin derivatives, mention may be made of those indicated in the Pierce catalog 1999-2000, pages 62 to 81, or in patent application WO 02/24754.

Idrabiotaparinux sodium;

Molecular Formula:C53H88N4O51S8.9NaCAS

Registry Number:405159-59-3

nonasodium methyl (2-deoxy-3 ,4-di-O-methyl-2-{6 – [5 - (2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl) pentanamido] hexanamido} -6-O-sulfo-α-D-glucopyranosyl) – (1 → 4) – (2,3-di-O-methyl-β-D-glucopyranosyluronate) – (1 → 4) – (2,3,6 -tri-O-sulfo-α-D-glucopyranoside) – (1 → 4) – (2,3-di-O-methyl-α-L-idopyranosyluronate) – (1 → 4) -2,3,6 – tri-O-sulfo-α-D-glucopyranoside

•  抗血栓治療中の出血率を低下させるためのイドラビオタパリナックスの使用

………………..

SYNTHESIS

WO2002024754A1

FIGURE 9

Synthesis of the pentasaccharide 39 

39

PREPARATION 34

Methyl _{(6-O-acetyl-2-azido-2-deoxy-3)} 4-di-0-methyl-O-glucopyranosyl) – (1 → 4) – (benzyl 2,3-di-O-methyl- β-D-glucopyranosyluronate) – (1 – → 4) – (3,6-di-O-acetyl-2-0-benzyl–D-glucopyranosyl) – (1 -> 4) – (benzyl 2,3 -di-O-methyl-a-idopyranosyl-uronate) – (1 → 4) -2,3,6-tri-0-benzyLa-D-glucopyranoside (39)

Compound 6-0-acetyl-2 was dissolved -azido-2-deoxy-3 ,4-di-0-methyl-, β-D-glucopyranose trichloroacetimidate 38 (265 mg, 0.631 mmol) (obtained by J. Basten, and Chem. Lett Bioorg. Med. al.. (1992), 2 (9), 901)

and

compound 32 (584 mg, 0.420 mmol) (obtained by P. Westerduin and Med. Bioorg Chem. al., 1994, 2, 1267) in a dichloromethane / diethyl ether 1/2 (v / v) (28.5 mL).

After addition of 4 Å molecular sieves powder, the mixture is cooled to -20 ° C. and a 0.1 M solution of trimethylsilyl trifluoromethanesulfonate in dichloromethane (94.6 uL). After 10 minutes, again added the imidate (53.8 mg) and a 0.1 M solution of trimethylsilyl trifluoromethanesulfonate in dichloromethane (19.2 uL). After 10 minutes, the mixture was neutralized by addition of solid sodium hydrogen carbonate. After filtration and concentration, the residue was purified by column chromatography on silica gel (toluene / ethyl acetate 3/1 (v / v)) to give 499 mg of compound 39. [Α] = +66 (c = 1, 07, dichloromethane).

FIGURE 10 -Summary of the pentasaccharide 44 (Method I)

COMPOUND 39

COMPD 40

COMPD 44

……………………

US20120232262

In scheme 1, the starting, intermediate and final compounds are the following:

• • compound (I): N-succinimidyl N-biotinyl-6-aminocaproate,
  • compound (II): N-biotinyl-6-aminocaproic acid,
  • compound (II′): N-biotinyl-6-aminocaproate carboxylate,
  • compound (III): biotin,
  • compound (III′): cyanomethyl biotinate.

EXAMPLE 1 Preparation of the Compound (I)

The reactions are monitored by LC with the following conditions: Symmetry C18 150×4.6 mm 5μ column (Waters); eluent A: 0.01 M KH₂PO₄ buffer adjusted to pH=3; eluent B: acetonitrile; flow rate 1 ml/min; gradient: t=0 min A/B 85/15, t=9 min A/B 65/35, t=10 min A/B 85/15, t=15 min A/B 85/15. This method makes it possible to visualize the biotin (compound (III), tR=4.5 min), the intermediate activated ester (III′) (tR=8.4 min), the N-biotinyl-6-aminocaproic acid (compound (II), tR=5.5 to 5.6 min), the intermediate mixed anhydride (II′) (tR=11.2 min) and the N-succinimidyl N-biotinyl-6-aminocaproate (compound (I), tR=7.9 to 8.2 min).

1.1: Preparation of the Compound (II)

7.5 kg of biotin (III), triethylamine (15 l, 2 V, 3.5 eq), NMP (15 l, 2 V) and, finally, chloroacetonitrile (3.5 kg, 0.47 OU, 1.5 eq) are charged to a reactor. The medium is heated to 60° C. After this temperature has been maintained for 2 h, an LC analysis shows that all the biotin has been converted into compound (III′) (<2%). The medium is cooled to 50° C. and then transferred into another reactor, containing aminocaproic acid (9.05 kg, 1.206 OU, 2.2 eq). Rinsing is carried out with NMP (0.1 V). The medium is heated to 100° C. and maintained at this temperature for 2 h. An LC analysis shows that less than 2% of activated biotin (III′) remains. The medium is cooled to 60° C. Acetonitrile (60 l, 8 V) preheated to 55° C. is run in. The mixture is stirred for 30 minutes at 60° C., and then cooled to 20° C. Stirring is carried out for 1 h. The suspension is filtered, then rinsing is carried out with 3 times acetonitrile (5 V) and then with THF (5 V). Drying is carried out under vacuum at a maximum of 60° C. until there is no change in weight. 12.0 kg of the compound (II) are thus obtained, with a yield of 109% and an organic purity, measured by LC, of 98.6%.

10.0 kg of the compound (II) are recharged to a reactor. Hydrochloric acid (90 l, 9 V of water+10 l, 1 V of 36% HCl) is then added. The suspension is stirred at 20° C. for 30 min. The suspension is filtered and rinsing is carried out 3 times with water (4 V, 40 l), then twice with THF (3.5 V). Drying is carried out under vacuum at a maximum of 45° C. until there is no change in weight. 6.1 kg of the compound (II) are thus obtained, with a yield of 66%.

1.2: Preparation of the Compound (I)

In a reactor, 3 kg of the compound (II) are suspended in DMF (25 l, 8.3 V) and the temperature is brought to −5° C. Triethylamine (1.02 kg, 0.34 OU, 1.2 eq) is then added. After stirring for 15 minutes, ethyl chloroformate (1.1 kg, 0.365 OU, 1.2 eq) is added gently (over the course of at least 1 h). Rinsing is carried out with DMF (0.9 l, 0.3 V). The medium is stirred at −5° C. for at least 2 h. The suspension becomes finer and yellow. An LC analysis shows that all the compound (II) (<3%) has reacted.

N-Hydroxysuccinimide (1.04 kg, 0.386 OU, 1.2 eq) in solution in DMF (3 l, 1 V) is then introduced in 1 step (over the course of at least 20 min). Rinsing is carried out with DMF (1.5 l, 0.5 V). The medium is stirred for 1 h 30 at −5° C. An LC analysis shows that the presence of residual compound (II) is less than 3%. The temperature is brought to 22° C., the suspension is taken up in DCM (12 V, 36 l) and the resulting organic phase is washed with water (15 l, 5 V). The organic phase is drawn off and the aqueous phase is extracted twice with DCM (30 l, 3 V). The organic phases are mixed and are washed with water (1.5 l, 0.5 V). The organic phase is concentrated to 6 V, i.e. 181. Heating is carried out at 40° C. and MTBE (6.25 V, 19 l) is added over the course of a minimum of 1 h. The mixture is maintained at 40° C. for 1 h, and then MTBE (8.75 V, 26 l) is added over the course of a minimum of 2 h. The mixture is maintained at 40° C. for at least 30 minutes, and then cooled to 20° C. over the course of a minimum of 2 h, and maintained at this temperature for 30 minutes. The suspension is filtered by suction and the cake is washed with acetone (5 V, 15 l) and then twice more with acetone (2 V, 6 l). The resulting product is filtered by suction and dried in an oven under vacuum at a maximum of 40° C. until there is no change in weight.

3 kg of the compound (I) are thus obtained in the form of a cream powder, with a yield of 80% and an organic purity, measured by LC, of 96.0%. Except for the compound (II), the presence of which is not problematic for a subsequent coupling reaction with a polysaccharide since it will be inert during this coupling, the purity of the compound (I) is 98%.

The biotinylated polysaccharides, the preparation of which is described above, are for example such as those described in patent applications WO 02/24754 and WO 2006/030104. They may in particular be the biotinylated pentasaccharide known under the International Nonproprietary Name “idrabiotaparinux” and described in patent application WO 02/24754, or the biotinylated hexadecasaccharides described in examples 1 and 2 of patent application WO 2006/030104.

In order to prepare these biotinylated polysaccharides, the compound (I) is coupled, respectively, with the pentasaccharide 44 described in patent application WO 02/24754

pentasaccharide 44: methyl (2-amino-2-deoxy-3,4-di-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-β-D-glucopyranosyluronic acid)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronic acid)-(1→4)-2,3,6-tri-O-sulfonato-α-D-glucopyranoside

EXAMPLE 2

Preparation of a biotinylated polysaccharide, idrabiotaparinux

A solution of 1.22 kg of the crude pentasaccharide 44 (containing salts), as described in patent application WO 02/24754, is prepared in 8.51 of water (7 V). 0.5 kg (1.6 eq) of the compound (I), 0.12 kg (2.0 eq) of NaHCO₃ and 0.37 kg of NaCl are added thereto. The solution is in the form of a white suspension. 3.7 l of acetone are added thereto and the reaction medium is stirred at approximately 25° C. for at least 22 h. This suspension is then slowly run into a mixture of ethanol (120 l) and MTBE (60 l) cooled beforehand to approximately 4° C., which makes it possible to precipitate the biotinylated pentasaccharide. The resulting suspension is then filtered and rinsed successively with absolute ethanol and acetone. The precipitate is oven-dried under a vacuum until there is no change in weight. 1.60 kg of crude idrabiotaparinux (containing salts) are thus obtained in the form of a cream powder, with an organic purity of 99%, and with a yield of 109% with respect to the pentasaccharide 44 and a chemical yield of 70% over the last 3 stages.

………………………….

WO2010023374A1

Compound of Preparation Example 1:

Methyl (2 – [N-(6-aminohexanoyl)]-2-deoxy-3 ,4-di-O-methyl-6-O-sulfonato-UD-glucopyranosyl) – (1 → 4) – (acid 2, 3 -di-O-methyl-β-D-glucopyranosyluronic) – (1-rf) – (2,3,6-tri-O-sulphonate-D-glucopyranosyl) – (1 → 4) – (2,3 – di-O-methyl-alpha-L-idopyranosyluronique) – (1 → 4) -2,3,6-tri-Osulfonato-D-glucopyranoside, sodium salt

Compound 1

1) Preparation of 6 – (benzyloxycarbonylamino) hexanoate succinimidyl

To a solution of 6 – (benzyloxycarbonyl amino) hexanoic acid (1.00 g, 3.77 mmol) in dimethylformamide (20 mL) was added triethylamine (0.63 mL, 4.52 mmol) and stirring the mixture at room temperature under argon for 30 minutes. The solution was cooled to ^{0 °} C and added dropwise ethyl chloroformate (0.43 mL, 4.52 mmol). After two hours at room temperature, N-hydroxysuccinimide (0.52 g, 4.52 mmol) and stirring the mixture overnight at room temperature. Evaporated to dryness before the residue in water to which is added with ethyl acetate. The phases were separated and the aqueous phase is extracted with ethyl acetate. The organic phases are combined, dried over sodium sulfate, filtered and evaporated to dryness before purification on a column of silica gel with pentane mixture of ethyl acetate / (75/25 v / v) as eluent. Once the fractions evaporated to give 1.13 g 6 – (benzyloxycarbonylamino) succinimidyl hexanoate as an oil. TLC: R _f = 0.22 on silica gel plate with a mixture of n-heptane/ethyl acetate (30/70 v / v) as eluent.

2) Preparation of compound the

Grafting the amine is carried out on the chain 44 pentasaccharide, or methyl (2 – amino-2-deoxy-3 ,4-di-0-methyl-6-0-sulfonato-α-D-glucopyranosyl) – (1 → 4) – (2,3 – di-0-methyl-β-D-glucopyranosyluronic) – (1 -> 4) – (2,3,6-tri-0-sulphonato-α-D-gluco-pyranosyl) – (1 → 4) – (2,3-di-O-methyl-α-L-idopyranosyl-uronic acid) – (1 → 4) – 2,3,6-tri-O-sulfo-α-nato- D-glucopyranoside, sodium salt, the preparation of which is described in patent application WO 02/24754:

44

To a solution of 6 – (benzyloxycarbonylamino) succinimidyl hexanoate (783 mg, 2.16 mmol) in N, N-dimethylformamide (10 mL) was added the pentasaccharide 44 (505 mg, 0.29 mmol). After stirring for 24 hours in an inert atmosphere and at room temperature, the solvent was evaporated under reduced pressure and the residue (40 mL) before washing the solution with chloroform (2 x 30 mL) dissolved in water. The chloroform phase is washed with water (10 mL) and aqueous phases were combined and evaporated to dryness under reduced pressure. The solid residue was triturated with 2-propanol (10 mL) and the suspension centrifuged for 5 minutes at 2500 rpm. The alcoholic phase is removed and replaced with 2-propanol (10 mL) and centrifugation was repeated. After having extracted the solvent, the crude product was dried under vacuum.

-2-deoxy-3 ,4-di-0-methyl-6-0-sulfonato-α-D [N-(benzyloxycarbonyl-6-aminohexanoyl)] – thus obtained 399 mg of the compound “, or methyl (2 -glucopyranosyl) – (1 → 4) - _(the acid 2 3-di-0-methyl-β-D-glucopyranosyluronic) – (1 → 4) – (2,3,6-tri-0-sulfonato-α- D-glucopyranosyl) – (1 – »4) – (2,3-di-0-methyl-α-L-idopyranosyluronique) – (1 – → 4) -2,3,6 – tri-O-sulphonate- α-D-glucopyranoside, wherein Pg is benzyloxycarbonyl:

January ¹ compound

Proton NMR at 200 MHz in deuterated water: The structure of the expected product is confirmed that the spectrum is identical to that performed on a product synthesized according to Example 5 of WO 02/24754 without the signals due to the biotin portion atoms but with signals of 7.4 to 7.5 ppm due to the benzyloxy group.

3) Preparation of compound 1

The product ‘s obtained at the end of the previous step (399 mg) is dissolved in deuterated water (10 mL). The solution of palladium on charcoal are treated with 10% (25 mg) and the solution was allowed to stir 20 hours in the presence of hydrogen at atmospheric pressure. Mixed with water (15 mL) was diluted, the catalyst was filtered and the solution was washed with chloroform (2 x 15 mL) before evaporating to dryness under reduced pressure. An aliquot (98 mg to 320 mg) of this product was purified on a column of Sephadex G-25 (2.5 x 50 cm) with water as eluent to give 25 mg of compound 1.

HPLC: Tr = 15.4 min column X-Terra RP-18 15W x 4.6 mm, 5μ particles of Waters in SA. With detection at 211 nm UV lamp.Eluent 1: water containing 0.02 M ammonium acetate and 0.05 M di-n-butylamine, adjusted to pH 7 with acetic acid. 2 Eluant: acetonitrile / water (90/10 v / v) containing 0.05 M di-n-butylamine and 0.08M acetic acid. The proportions of eluents are programmed so that the eluent composition is 10% 2 0 min. , 20% at 25 min. , 50% at 40 min. , 50% at 43 min. and 5% to 50 minutes. Proton NMR at 600 MHz in deuterated water: The structure of the expected product is confirmed that the spectrum is identical to that performed on a product synthesized according to Example 5 of WO 02/24754 without the signals due to the biotin portion atoms.

…………………………

IDRABIOTAPARINUX

REFERENCES

JOURNAL OF THROMBOSIS AND HAEMOSTASIS vol. 9, 2010, pages 92 – 99

BULLER HARRY R ET AL: “Idraparinux versus standard therapy for venous thromboembolic disease“, NEW ENGLAND JOURNAL OF MEDICINE, vol. 357, no. 11, September 2007 (2007-09) , pages 1094-1104,

EQUINOX INVESTIGATORS: “Efficacy and safety of once weekly subcutaneous idrabiotaparinux in the treatment of patients with symptomatic deep venous thrombosis.“, JOURNAL OF THROMBOSIS AND HAEMOSTASIS : JTH JAN 2011 LNKD- DOI:10.1111/J.1538-7836.2010.04100.X PUBMED:20946157, vol. 9, no. 1, January 2011 (2011-01), pages 92-99,

N. ENG. J. MED. vol. 357, 2007, pages 1094 – 104

PRANDONI P ET AL: “Idraparinux: review of its clinical efficacy and safety for prevention and treatment of thromboembolic disorders“, EXPERT OPINION ON INVESTIGATIONAL DRUGS, ASHLEY PUBLICATIONS LTD., LONDON, GB, vol. 17, no. 5, 1 May 2008 (2008-05-01), pages 773-777,

SAVI P ET AL: “Reversible biotinylated oligosaccharides: A new approach for a better management of anticoagulant therapy“, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, BLACKWELL PUBLISHING, OXFORD, GB, vol. 6, no. 10, 1 January 2008 (2008-01-01), pages 1697-1706,

Emerging anticoagulants.Kennedy B, Gargoum FS, Kennedy L, Khan F, Curran DR, O’Connor TM.Curr Med Chem. 2012;19(20):3388-416. Review.

PATENTS

Emblica officinalis

Triphala (/triːˈfɑːlə/ or /triːˈfælə/; Hindi/Sanskrit: त्रिफला, triphalā [trɪˈpʰɐlaː], “three fruits”)^[1] is an Ayurvedic^[2] herbal rasayana formula consisting of equal parts of three myrobalans, taken without seed: Amalaki (Emblica officinalis), Bibhitaki (Terminalia bellirica), and Haritaki (Terminalia chebula).^[1]

Medicinal use

In traditional Ayurvedic medicine, Triphala is used for:

• immune system stimulation^[3]
• improvement of digestion^[4][1]
• relief of constipation^[4][1]
• gastrointestinal tract cleansing^[4]
• relief of gas^[1]
• treatment of diabetes^[1]
• treatment of eye disease^[1]

These health claims have not been yet tested in clinical trials. Even within the practice of Ayurvedic medicine, there are controversies about the composition (amlaki, haritaki and bibhitaki), preparation, and medicinal uses of Triphala.^[5]

The active constituents are unknown. Triphala contains several compounds that have been proposed to be responsible for its claimed health benefits, including gallic acid, chebulagic acid, and chebulinic acid. ^[6][7]

Contemporary research on triphala

There is preliminary evidence that Triphala contains compounds with antioxidant properties in isolated cells and rats, however this has not yet been demonstrated in people.^{[6][8][9][10]}

Triphala, widely used by natural Ayurvedic healers in India for thousands of years, contains 3 different fruits: Harada, Amla and Bihara.  The word “Triphala”literally means “three fruits”.  The combination of these three fruits cleanses the gastro-intestinal tract in a natural and gentle way.  Basically our “bathroom experience” becomes a better one  That is the best way I can put it!!

Why should we cleanse?

It’s always a good idea to cleanse! Get rid of toxins that build up in our bodies so that our bodies can function most efficiently and have that bright glowing skin we all crave and want!  More energy and feel less bloated!

And I’m not talking about cleansing with juicing or not eating.  No no, that’s a whole other conversation.  I absolutely believe in still eating a healthy diet while “cleansing”/taking Triphala.

I have suggested Triphala to many clients, students and friends and all of them have seen results.  You can call it a form of laxative if you’d like but this is totally safe and gentle on the body.  Yes, we are all different but seems like this one might be a miracle worker and work for everyone!

Suggested use: Take one pill before bedtime.  *Take on and off for a period of time OR once in a while when you feel you need it.  I usually take it when I feel I need a cleanse- about one or two times a week (usually when I have consumed a bigger meal or more food than usual).

Benefits of Triphala:

• detoxify and cleanses the colon of toxins
• removes excess fats
• purifies the blood
• removes toxins from the liver
• reduces some forms of cholesterol (serum cholesterol)
• reduces high blood pressure
• high nutritional value: including high levels of vitamin C
• high in antioxidants
• strengthens hair roots and enriches hair color

The three fruits contained in Triphala are

Amalaki (Indian Gooseberry),

Haritaki (Indian Gallnut or Terminalia chebula),

and Bibhitaki (Beleric Myrobalan or Terminalia bellerica).

The prokinetic cleanser

An immensely popular Ayurvedic herbal formula,Triphala(Terminalia chebula,Terminalia bellirica and Emblica officinalis) is an effective bowel cleanser. It combines the goodness of Indian Gooseberry, Belleric Myrobalan and Chebulic Myrobalan, which work together to produce effective bowel movements.

The herbal compound provides overall support for digestion and helps ensure that the digestive tract works at optimal levels. Triphala relieves constipation and regularizes the digestive system, without disrupting the fluid-electrolyte balance in the body.

The herbs that make up Triphala are found in abundance in India.

Triphala, the well-known traditional Ayurvedic formulation, makes an excellent skin tonic. It is one of the most popular Ayurvedic medicinal herbs, prescribed by a number of Ayurvedic practitioners. Triphala literally means ‘three fruits’. The three fruits contained in Triphala are Amalaki (Indian Gooseberry), Haritaki (Indian Gallnut or Terminalia chebula), and Bibhitaki (Beleric Myrobalan or Terminalia bellerica). Since Triphala is tridoshic – equally balancing for Vata, Pitta and Kapha – it is beneficial for all skin types. Triphala nourishes the skin, both directly and indirectly. Amla (Indian gooseberry), one of the three ingredients in Triphala, is the richest known natural source of Vitamin C. Apart from the rich source of Vitamin C, Triphala also contains calcium – an important nutrient that helps enhance skin clarity and brings dull, tired skin to life.

Preparation Of Triphala Rasayana
Triphala Rasayana is usually prepared by mixing triphala with equal quantity of madhuka (mahua tree), tavakshir (East Indian arrowroot) pippali (long pepper), saindhava (long salt), and each one of the loha (iron), suvarna (gold), vacha (Acorus calamus) with either honey, ghee or sugar, in equal quantity.

Benefits Of Triphala
Triphala Rasayana is beneficial is promoting ojas, the finest product of digestion that prevents the occurrence of many diseases, creates luster and make the skin exude its natural glow and radiance.
It nourishes both the body and the mind, thereby promoting longevity of life. Therefore, Triphala Rasayana is very much beneficial for adults and children alike.
The Rasayana is especially beneficial for eyes. In case one has problems in eye sight, opting for Triphala Rasayana would be the best bet.
The Rasayana creates a balance in the cholesterol level, by removing ama from the fat tissue.
It helps in the purification of urinary tract, thereby helping the prevention of urinary tract diseases.
It also strengthens and cleanses the liver, which is one of its main functions. This ensures that the liver, one of the important parts of the body, stays healthy. It can also be said that the consumption of Rasayana prevents diseases related to the functioning of liver.
The medicine also helps the management of weight. Thus, it is beneficial for people, who want to loose weight.
It enhances the thirteen agnis (digestive fires), especially the main digestive fire in the stomach.
Triphala Rasayana is helpful in pacifying Kapha and Pitta. If taken on a regular basis, the Rasayana can be a powerful anti-aging medicine.
People suffering from skin inflammation, heat, infection, obesity will find the consumption of Triphala Rasayana as beneficial.
Diseases such as fatigue and anemia can be effectively cured by the regular consumption of Triphala Rasayana, if taken according to the prescribed doses.

Garden Cress Extract Kills 97% of Breast Cancer Cells in Vitro: Garden cress, like broccoli, is a cruciferous-family vegetable but is unique because it contains very high amounts of BITC (benzyl isothiocyanate) which has emerged as a powerful anti-cancer compound. In this study, BITC was seen to kill 97% of ER- breast‪cancer‬ cells (MDA-MB-231) after 24 hours of treatment. For comparison, the same dose of sulforaphane from ‪‎broccoli‬ killed only 75% of the cancer cells.

In other research, BITC has been found to slow the rate of breast cancer metastasizing by 86% and when given to mice, resulted in breast tumors 53% smaller than in untreated mice. BITC is now being intensively studied for a variety of cancers and has been shown in lab studies to be active against melanoma, glioma, prostate cancer, lung cancer, ovarian cancer, pancreatic cancer and others. Garden ‪‎cress‬ is one of the best sources of BITC. Other good sources include cabbage, Indian cress, Japanese radish (in particular Karami daikon) and, quite surprisingly, papaya seeds. As with other‪cruciferous‬ vegetables, the best way to eat cress is raw in order to maximize the delivery of BITC.

http://www.ncbi.nlm.nih.gov/pubmed/17121941

http://extension.usu.edu/files/publications/publication/HG_Garden_2006-05.pdf

and

http://nopr.niscair.res.in/bitstream/123456789/12732/1/IJNPR%202(3)%20292-297.pdf

BITC

Botanical name: Lepidium sativum L.
Family: Brassicaceae = Cruciferae
Common names. English: cress, common cress, garden cress, land cress, pepper cress; Spanish: mastuerzo, mastuerzo hortense, lepidio, berro de jardín (Spain), berro de sierra, berro hortense (Argentina), escobilla (Costa Rica); Catalan: morritort, morrisà, Portuguese and Galician: masturco, mastruco, agrião-mouro, herba do esforzo; Portuguese: mastruco do Sul, agrião (Brazil); Basque: buminka, beatzecrexu

Synonyms/Common Names/Related Substances:

• Alpha-linolenic acid (ALA), agrião (Portuguese), agrião-mouro (Portuguese, Galician), beatzecrexu (Basque), berro de jardín (Spanish), berro de tierra (Spanish), berro hortense (Spanish), benzyl isothiocyanate (BITC), Brassicaceae (family), bran, buminka (Basque), common cress, cress, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), escobilla (Spanish), endosperm, fiber, garden cress seed oil (GCO), garden pepper grass, glucosinolates, glutamic acid, herba do esforzo (Portuguese, Galician), hurf (Arabic), indoles, isothiocyanates, kardamon (Greek), land cress, linoleic acid (LA), lectin, lepidio (Spanish), Lepidium sativium, Lepidium sativum, leucine, mastruco (Portuguese, Galician), mastruco do sul (Portuguese), mastuerzo (Spanish), mastuerzo hortense (Spanish), methanol, morrisá (Catalan), morritort (Catalan), nasturtium (Latin), nasum torcere (Latin), omega-3 fatty acid, pepper cress, pepper grass, pepperwort, sulforaphane, tuffa’ (Arabic), turehtezuk (Persian), water cress, whole meal.
• Combination product example: SulforaWhite (a liposomal preparation that contains Lepidium sativum sprout extract, glycerin, lecithin, phenoxyethanol, and water).

Garden cress [commonly known as aliv in Marathi or halim in Hindi] is a green, cool-season perennial plant used as a leafy vegetable, typically used as a garnish. Undisturbed, the plant can grow to a height of two feet with minimal maintenance. When mature, garden cress produces white or light-pink flowers, and small seed pods. It has long leaves at the bottom of the stem and small, bright-green, feather-like leaves arranged on opposite sides of its stalks at the top.

Garden Cress, also called Pepper Wort, is an herb that is botanically known as Lepidium Sativum. It is referred to as ‘Aliv’ in Marathi and ‘Halim’ in Hindi. Belonging to the family Cruciferae, it is grown in all parts of India and is often used in the Indian cuisine. The leaves, roots, as well as seeds of this plant are used in cooking as they are extremely nutritious and also therapeutic in nature. The flowers of this plant are either white or light-pink in color.

This herb is the best source of iron and is hence recommended in the treatment of iron-deficiency anemia. It is also rich in folate, calcium, ascorbic acid, tocopherol, and beta-carotene. Garden Cress seeds are loaded with not just protein, but also linoleic and arachidic fatty acids. Since they contain phytochemicals that mimic estrogen to some extent, intake of these seeds is known to regulate menstruation and stimulate milk production in lactating mothers. That is precisely why women are given foods containing Garden Cress following childbirth.

The blood-purifying as well as antioxidant properties of this amazing plant are well documented. Hence, its regular consumption can greatly help to boost one’s immunity and prevent a gamut of diseases. It acts as a general tonic and can also help to increase the libido naturally. Since the testae of these seeds contain mucilage, they are invaluable in the management of both dysentery and constipation. The whole plant, along with its seeds, is said to be good for the eyes too. Hence, it is advisable to add it raw to salads, sandwiches, and chutneys, or to simply use it as a garnish along with coriander leaves for any food item.

Pregnant women should avoid taking Garden Cress in any form because it has the ability to induce uterine contractions and thereby trigger a spontaneous abortion. Also, since it is goitrogenic in nature, it may not be suitable for patients suffering from hypothyroidism. The oil derived from Garden Cress seeds is edible and can therefore be used as a cooking medium; however, some people may experience symptoms of indigestion due to its use. Such individuals should discontinue using this oil or mix it with some other edible oil, so as to dilute it and reduce its adverse effects.

Cress (Lepidium sativum), sometimes referred to as garden cress to distinguish it from similar plants also referred to as cress, is a rather fast-growing, edible herb. Garden cress is genetically related to watercress and mustard, sharing their peppery, tangy flavor and aroma. In some regions, garden cress is known as mustard and cress, garden pepper cress, pepperwort pepper grass, or poor man’s pepper.^[1][2]

This annual plant can reach a height of 60 cm (~24 inches), with many branches on the upper part. The white to pinkish flowers are only 2 mm (1/12 of an inch) across, clustered in branched racemes.^[3][4]

Origin of the name

Cultivation of this species, which is native to Southwest Asia (perhaps Persia) and which spread many centuries ago to western Europe, is very old, as is shown by the philological trace of its names in different Indo-European languages. These include the Persian word turehtezuk, the Greek kardamon, the Latin nasturtium and Arabic tuffa’ and hurf. In some languages there is a degree of confusion with watercress. It seems that the meaning of the word nasturtium (nasum torcere, because its smell causes the nose to turn up) must have been applied initially to garden cress, as both Pliny and Isidoro de Sevilla explain. The confusion remains with the terms used by the Hispano-Arabs. The word hurf is applied without distinction to watercress and garden cress (several species certainly of up to three different genera: Nasturtium, Lepidium and Cardaria). Thus the medieval agronomists of Andalusia went as far as differentiating between several hurf, such as hurf abyad, hurf babili, hurf madani….

Garden cress in agriculture

Garden cress is commercially grown in England, France, the Netherlands and Scandinavia.^[5]

Cultivation of garden cress is practical on both mass scales and on the individual scale. Garden cress is suitable for hydroponic cultivation and thrives in slightly alkaline water. In many local markets, the demand for hydroponically grown cress can exceed available supply, partially because cress leaves are not suitable for distribution in dried form, so can be only partially preserved. Consumers commonly acquire cress as seeds or (in Europe) from markets as boxes of young live shoots.^[5]

Edible shoots are typically harvested in one to two weeks after planting, when they are 5–13 cm (2 – 5 inches) tall.^[6]

Properties, uses and cultivation

Xenophon (400 _BC) mentions that the Persians used to eat this plant even before bread was known. It was also familiar to the Egyptians and was very much appreciated by the Greeks and Romans, who were very fond of banquets rich in spices and spicy salads. Columela (first century) makes direct reference to the cultivation of garden cress. In Los doce libros de Agricultura, he writes: ” …immediately after the calends of January, garden cress is sown out… when you have transplanted it before the calends of March, you will be able to harvest it like chives, but less often… it must not be cut after the calends of November because it dies from frosts, but can resist for two years if it is hoed and manured carefully… there are also many sites where it lives for up to ten years” (Book XI). The latter statements seem to indicate that he is also speaking of the perennial species L. Iatifolium, as L. sativum is an annual.

Almost all of the Andalusian agronomists of the Middle Ages (Ibn Hayyay, Ibn Wafid, Ibn al-Baytar, Ibn Luyun, Ibn al-Awwam) and many of the doctors, such as Maimonides, mention garden cress. Ibn al-Awwam also includes references from Abu al-Jair, Abu Abdalah as well as from Nabataean agriculture and, among other comments, he says: “Garden cress is sown between February and April (in January in Seville). It has small seeds which are mixed with earth for sowing to prevent the wind carrying them away…. It is harvested in May and is grown between ridges, in combination/conjunction with flax cultivation.”

Many of the authors of the old oriental and Mediterranean cultures emphasized the medicinal properties of cress, especially as an antiscorbutic, depurative and stimulant. Columela notes its vermifugal powers. Ibn al-Awwam refers to certain apparently antihistaminic properties, since it was used against insect bites and also as an insect repellent, in the form of a fumigant. It was perhaps Ibn al-Baytar, an Andalusian botanist (eighth century), who collected most information on its properties, summarizing the opinions of other authors such as El Farcy, who says that it incites coitus and stimulates the appetite; Ibn Massa, according to whom it dissipates colic and gets rid of tapeworms and other intestinal worms; or Ibn Massouih, who mentions that it eliminates viscous humours. Ibn al-Baytar also says that it is administered against leprosy, is useful for renal “cooling” and that, if hair is washed with garden cress water, it is “purified” and any loss is arrested.

In Iran and Morocco, the seeds are used as an aphrodisiac. In former Abyssinia, an edible oil was obtained from the seeds. In Eritrea, it was used as a dyestuff plant. Some Arab scholars have attributed garden cress’s reputation among Muslims to the fact that it was directly recommended by the Prophet.

Garden cress’s main use was always as an aromatic and slightly pungent plant. Not only in antiquity but also in the Middle Ages it enjoyed considerable prestige on royal tables. The young leaves were used for salads. The ancient Spartans ate them with bread. This use still continues and they are also eaten with bread and butter or with bread to which lemon, vinegar or sugar is added. However, it is mainly used nowadays in the seedling stage, the succulent hypocotyls being added to salads and as a garnish and decoration for dishes.

The roots, seeds and leaves have been used as a spicy condiment. Columela explains how oxygala, a type of curd cheese with herbs, was prepared: “Some people, after collecting cultivated or even wild garden cress, dry it in the shade and then, after removing the stem, add its leaves to brine, squeezing them and placing them in milk without any other seasoning, and adding the amount of salt they consider sufficient…. Others mix fresh leaves of cultivated cress with sweetened milk in a pot…”.

L. Iatifolium L. stands out for its horticultural interest; although it grows spontaneously on the edges of rivers and lakes, it is also occasionally grown in the same way as L. sativum. Its young leaves can be used for salads; the ancient Greeks and Romans used to grow it for this purpose. Its leaves and seeds were also used as a spicy condiment. Several sauces are prepared with its leaves, including in particular the bitter sauce of the paschal lamb of the Jews. The seeds of this species were known in England as the poor people’s pepper. The roots have been used on occasion as a substitute for radish.

In the fifteenth century, we know through Alonso de Herrera that garden cress was one of the vegetables most widely eaten in Castile. During the sixteenth century, obstinate attempts were made to introduce it into America. Right up to the beginning of the nineteenth century, its cultivation in Spain continued to be important, since Boutelou and Boutelou (1801) deal specifically with this crop in their Tratado de la huerta, commenting on the existence of several cultivars. At present, the cultivation of cress is very occasional in countries such as Spain and France. Water cress, in competition with garden cress, has eclipsed the cultivation of the latter. However, this is not the case in other central European countries or the United Kingdom, where its use is normal and the system of cultivation has changed substantially.

Botanical description

Cress is an annual, erect herbaceous plant, growing up to 50 cm. The basal leaves have long petioles and are lyrate-pinnatipartite; the caulinar leaves are laciniate-pinnate while the upper leaves are entire. The inflorescences are in dense racemes. The flowers have white or slightly pink petals, measuring 2 mm. The siliquae measure 5 to 6 x 4 mm, are elliptical, elate from the upper half, and glabrous. Cress flowers in the wild state between March and June.

It is an allogamous plant with self-compatible and self-incompatible forms and with various degrees of tolerance to prolonged autogamy. There are diploid forms, 2n = 2x = 16, and tetraploid forms, 2n = 4x =32. A degree of variability is noted in the character of the basal leaves which are cleft or split to a greater or lesser degree, a character which is controlled by a single incompletely dominant gene.

Ecology and phytogeography

Cress is a plant that is well suited to all soils and climates, although it does not tolerate frosts. In temperate conditions, it has a very rapid growth rate. It grows subspontaneously in areas transformed by humans, close to crops or human settlements. It appears in this way on the Iberian peninsula, mainly in the eastern regions.

Wild cress extends from the Sudan to the Himalayas. Most authors consider it to be a native of western Asia, whence it passed very quickly to Europe and the rest of Asia as a secondary crop, probably associated with cultivars of flax. Vavilov considers its main centre to be Ethiopia, where he found the widest variability; the Near East, central Asia and the Mediterranean are considered secondary centres. It is now naturalized in numerous parts of Europe, including the British Isles.

Cress in cookery

Genetic diversity

The genus Lepidium is made up of about 150 species, distributed throughout almost all temperate and subtropical regions of the world. On the Iberian peninsula and the Balearic Islands, at least 20 species or subspecies exist among the autochthonous and allochthonous taxa, some genetically close to L. sativum. Seven of them are exclusively endemic to the peninsula or, at the very most, are common with North Africa. Other close species are L. campestre (L.) R. Br. and L. ruderale L. which also have edible leaves. The leaves of L. campestre are used to prepare excellent sauces for fish.

Common cress (L. sativum L.), with regard to the anatomy of the leaf, stem and root, has been divided into three botanical varieties: vulgare, crispum and latifolium. The latter is the most mesomorphic, crispum the most xeromorphic and vulgare intermediate.

At present, most of the studies on the variability and development of new cultivars are being carried out in liaison with the VIR of St Petersburg, where there is a good collection of material. Of the 350 forms of garden cress studied in the Ukraine, Uzkolistnyti 3 was the best, being highly productive and of good quality. It is being used as the basis of improvement programmes, as it appreciably surpasses the best Soviet varieties in production and quality. Other cultivars well suited to European Russia are Tuikers Grootbladige (broad-leaved) and the lines Mestnyi k 137, k 106 and k 115. Of the types most cultivated in Europe, Early European, Eastern, Dagestan and Entire Leaved stand out, being distinguished by the length and shape of the leaf, earliness and susceptibility to cold. In Western Europe, one broad leaved type is especially appreciated (Broad Leaved French) as are curly types (Curly Leaved), the latter being used extensively to garnish dishes. In Africa, there are red, white and black varieties.

This crop is also arousing interest in Japan, and collecting expeditions to Nepal have been organized. Some specimens collected during an expedition to Iraq in 1986 are now stored in Abu Ghraib and in Gratersleben, Germany. There are also small collections of L. sativum in the PGRC in Addis Ababa (Ethiopia), at the ARARI of Izmir in Turkey and in Bari, Italy. At the Universidad Politécnica de Madrid there are accessions of 20 species of Lepidium, while the BGV of the Córdoba Botanical Garden keeps germplasm of the southern Iberian species of the genus.

Garden cress is added to soups, sandwiches and salads for its tangy flavor.^[6] It is also eaten as sprouts, and the fresh or dried seed pods can be used as a peppery seasoning (haloon).^[5] In England, cut cress shoots are commonly used in sandwiches with boiled eggs, mayonnaise and salt.

Garden cress can grow almost anywhere.

Nutrition profile

Garden cress is an important source of iron, folic acid, calcium, vitamins C, E and A. The seed contains arachidic and linoleic fatty acids. The seeds are high in calories and protein, whereas the leaves are an excellent source of vitamin A, C and folate.

Values for 100g of garden cress leaves

Energy	30 Kcal
Carbohydrates	5.5 g
Dietary fibre	1.1 g
Protein	2.6 g
Fat	0.7 g
Vitamin A	346 mcg
Folate	80 mcg
Vitamin C	69 mg
Calcium	81 mg
Iron	1.3 mg

Both the leaves and stems of cress can be eaten raw in salads or sandwiches, and are sometimes called cress sprouts. When buying cress, look for firm, evenly coloured, rich green leaves. Avoid cress with any signs of slime, wilting, or discoloration. If stored in plastic, it can last up to five days in a refrigerator. To prolong the life of cress, place the stems in a glass container with water and cover them, refrigerating the cress until it is needed.

Cultivation practices

Cress is an easily grown plant with few requirements. It can be broadcast after the winter frosts or throughout the year in temperate climates. However, Boutelou and Boutelou (1801) were already recommending sowing in shallow furrows, which enables surplus plants to be thinned out and facilitates hoeing. Sowing has to be repeated every 15 to 20 days so that there is no shortage of young shoots and new leaves for salads – the leaves of earlier sowings begin to get tough and are no longer usable. The seed sprouts four or six days after sowing, depending on the season, and the leaves are ready for consumption after two or three weeks.

The usual form of cultivation continues to be as described, with 15 to 20 cm between rows and the use of irrigation in the summer, since they are lightly rooted seedlings which can dry up in a few days. Its growth is very rapid and harvesting can begin in the same month as sowing, with yields reaching 6 tonnes per hectare.

Health benefits of garden cress

For women’s health

Emenagogue: Garden cress has mild oestrogenic properties. It helps to regulate the menstrual cycle.

Galactogogue: Kheer made of garden cress seeds increases milk production and secretion in lactating mothers. Because of its high iron and protein content, it is often given post-partum to lactating mothers.

Aphrodisiac: Garden cress helps to improve libido.

For the gastro intestinal tract

Garden cress helps purify blood and stimulate appetite. It is used during constipation as a laxative and a purgative. Paste made of the seeds can be taken internally with honey to treat amoebic dysentery. The mucilage of the germinating seeds allays the irritation of the intestines in dysentery and diarrhoea. Garden cress crushed and drunk with hot water is beneficial to treat colic especially in infants.

For the respiratory tract

Garden cress seeds are good expectorants and when chewed they treat sore throat, cough, asthma and headache. The aerial parts are used in the treatment of asthma and cough.

For anaemia

Garden cress seeds being the richest source of non-haeme iron [iron found in haemoglobin which is an easily absorbed dietary iron.] help to increase the haemoglobin levels. When taken regularly, it helps to alleviate anaemia. It is advisable to have vitamin C half an hour after consumption of these seeds as it enhances iron absorption.

For diabetes

The seed coat of germinating seeds contains mucilage, which has a phytochemical called lepidimoide. Studies show that seeds of the plant lower the glycemic response to a test meal.

note  sodium 2-O-rhamnopyranosyl-4-deoxy-threo-hex-4-enopyranosiduronate (designated lepidimoide)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1075667/

epi-Lepidimoide

Sodium 6-deoxy-2-O-(4-deoxy-β-L-threo-hex-4-enopyranuronosyl)-α-L-glucopyranose

cas 145039-76-5 and 157676-09-0

The total synthesis of the unsaturated disaccharide, lepidimoide 4-deoxy--l-threo-hex-4-enopyranuronosyl-(1->2)-l-rhamnopyranose sodium salt, has been carried out from d-glucose and l-rhamnose (Tetrahedron Lett. 1993, 34, 2653), but the process is very long and complicated. A method for more easily producing this compound and in large quantities is necessary for further research. We have succeeded in conveniently synthesizing lepidimoide from okra (Hibiscus esculentus L.) fruit mucilage. At the same time, the isomer (epi-lepidimoide) was obtained as a byproduct. The structure was determined as the 4-deoxy--l-threo-hex-4-enopyranuronosyl-(1->2)-6-deoxy-l-glucopyranose sodium salt by spectral analysis. We found that lepidimoide easily epimerized to epi-lepidimoide in alkaline media. Both lepidimoide and epi-lepidimoide exhibited the same high activity in the cockscomb hypocotyls elongation test….Carbohydrate Research, Volume 339, Number 1, 2 January 2004 , pp. 9-19(11)

1. Dictionary of Natural Products, Supplement 3

   books.google.co.in/books?isbn=0412604302

   John Buckingham – 1996 – ‎Science

   Lepidimoide. L-30020. 6-Deoxy-2-0-(4-deoxy-fi-L-lhreo-hex-4-enopyranuronosyl)-L- mannose, 9CI [157676-09-0] HOA—O  …

   lepidimoide

   Sodium 2-O-L-rhamnopyranosyl-4-deoxy-alpha-L-threo-hex-4-eno-pyranosiduronate

   Molecular Formula: C₁₂H₁₇NaO₁₀   Molecular Weight: 344.247149

   sodium;(2S,3R,4S)-3,4-dihydroxy-2-[(2S,3R,4R,5R,6S)-2,4,5-trihydroxy-6-methyloxan-3-yl]oxy-3,4-dihydro-2H-pyran-6-carboxylate

   Sodium2-O-L-rhamnopyranosyl-4-deoxy-α-L-threo-hex-4-eno-pyranosiduronate

For cancer

Garden cress seeds contain antioxidants like vitamin A and E which help protect cells from damage by free radicals. Hence, these seeds have a chemoprotective [drugs which protect healthy tissue from the toxic effects of anticancer drugs] nature.

Anti-Cancer:

Being a family of Brassica family it has good anti cancer property. Garden cress seeds contain antioxidants like vitamin A and E which help protect cells from damage by free radicals. Hence, these seeds have a chemo protective nature.

Few years back garden cress seeds/ halim/ aliv was not a common food item or a familiar to be heard. But as years passed it’s popularity and it’s importance have been realized and now people are aware of some of the facts of these seeds. Though these facts are also accompanied by some myths. So I chose to write and clear few myths and doubts of these seeds so that maximum people can make use of it in their lives and improve quality of their diet and nutrition.

Nutritive value of these seeds is very high. It is available in almost all parts of the world. Its high nutritive value and cheaper availability makes it possible for people of all the sections of society to include in the diet and increase nutritive value of their meals. Garden cress seeds are very high in Iron and Folic acid content. These seeds are use as herbal medicine to treat iron deficiency anemia. People consuming 2tsp/day have seen to have good increased levels of hemoglobin over a period of 1-2 months. Garden cress seeds also contains calcium, ascorbic acid, tocopherol, and beta-carotene which helps to improve body’s immunity.Garden Cress seeds are loaded with not just protein, but also linoleic and arachidic fatty acids. Since they contain phytochemicals that resemble  estrogen to some extent, intake of these seeds helps to regulate menstruation and stimulate milk production in lactating mothers. That is  why women are given foods containing Garden Cress following childbirth.

Traditionally garden cress seeds were considered to be useful only during last few weeks of gestation and post delivery. It is considered to be hot food. But the truth is that these seeds have ability too increase uterine contraction. So in later stages of pregnancy it helps in inducing labour but if in case consumed in early stage of pregnancy (1st trimester) it leads to spontaneous abortion. It is also very carefully prescribed to a hypothyroid patients because it belongs to cruciferous family and is a goitrogen that prevent iodine absorption.

How to eat:

1. Roasted slightly with added salt.

2. Soaked in water then added to milk or juice.

3. Chikki or laddoo can be made. (preparation similar to til laddoo/chikki).

How much to eat:

Start with 1 tsp/day and then an be taken 1 tsp/2c a day.

Cress seeds have many more medicinal properties and researches are still on to find its benefits on health. Garden cress should be eaten in moderation. Excess consumption of these seeds may hv adverse effect on health.

For other things

Garden cress seeds are memory boosters because they contain arachidic and linoleic acids. They help gaining lean body mass because they are a good source of iron and protein. Research has proved that 60 per cent women have hair loss due to low iron levels and poor protein. A teaspoon of garden cress seeds soaked in lime water helps in iron absorption, which in turn strengthens hair. The plant is also used in treating bleeding piles. The leaves are mildly stimulant and diuretic, useful in scorbutic [related to or resembling scurvy] diseases and liver complaints. A paste of the seeds with water is applied to chapped lips, and against sunburn.

Side-effects

It is an abortifacient [substance that induces abortion], if had in excess. It contains goitrogens that prevent iodine absorption in thyroids and hence can lead to hypothyroidism. If large quantities of garden cress are consumed, the mustard oil it contains may cause digestive difficulties in some people who are sensitive to it. Therefore, garden cress should be eaten in moderation.

Other uses

Garden cress, known as chandrashoor, and the seeds, known as halloon^[7] in India, are commonly used in the system of Ayurveda to prevent postnatal complications.^{[citation needed]}

Garden cress seeds, since ancient times, have been used in local traditional medicine of India.^[8] Garden cress seeds are bitter, thermogenic, depurative, rubefacient, galactogogue, tonic, aphrodisiac, ophthalmic, antiscorbutic, antihistaminic and diuretic. They are useful in the treatment ofasthma, coughs with expectoration, poultices for sprains, leprosy, skin disease, dysentery, diarrhoea, splenomegaly, dyspepsia, lumbago, leucorrhoea, scurvy and seminal weakness. Seeds have been shown to reduce the symptoms of asthma and improve lung function in asthmatics.^[9]The seeds have been reported as possessing a hypoglycemic property^[10] and the seed mucilage is used as a substitute for gum arabic and tragacanth.

Cress may be given to budgerigars.^[11] The seeds are employed as poultice for removing pain, swelling etc.Some use it in the belief that it can cure asthma, bronchitis bleeding piles.^[12]

Some use Lepidium sativum seeds for indigestion and constipation.^[13]

Prospects for improvement

Most of the genetic improvement work on garden cress is being carried out in the CIS, with little or no work being done at present in the countries of western Europe. Mainly early cultivars with a prolonged production period and better cold tolerance are being developed.

Cress can be grown and used like white mustard. It germinates more slowly at low temperatures, the emergence period being three or four days longer. Shortening this period is an interesting improvement objective.

However, cress’s recovery and its greater presence on markets mainly depends on a modification of cultivation and marketing techniques. In countries such as the United Kingdom, where this vegetable is normally to be found at the markets, cultivation takes place in greenhouses throughout the year. The whole succulent hypocotyls of the very young seedlings are eaten. The seed is placed on the soil surface on soft, level beds. It is finely sprinkled with water and then covered with sackcloth which has been steam-sterilized and moistened. The latter is frequently wetted to maintain moisture and is removed when the seedlings reach 4 to 5 cm in height (after approximately seven days in spring and autumn and ten days in winter). The yellowish leaves turn green after two to three days.

The cress is harvested when the first pair of cotyledon leaves have developed and it is marketed in small bags or trays, sometimes together with seedlings of white mustard.

Garden cress and white pepper are sometimes sown in the plastic trays or bags in which they will be sold, generally in peat with a nutrient solution.

References

• Time-lapse video showing garden cress growth

ASPARAGUS

Asparagusic acid

Asparagusic acid is the organosulfur with the formula S₂(CH₂)₂CHCO₂H. The molecule contains both carboxylic acid and disulfide functional groups. It is present in the vegetable asparagus and may be the metabolic precursor to other odorous thiol compounds.

The material was originally isolated from an aqueous extract of asparagus.

Biosynthetic studies revealed that asparagusic acid is derived from isobutyric acid. This colorless solid has a melting point (m.p.) of 75.7–76.5 °C. The corresponding dithiol (m.p. 59.5–60.5 °C) is also known; it is called dihydroasparagusic acid or dimercaptoisobutyric acid.

3D MODEL

Over the past forty years several papers have been published on the subject, and several studies undertaken, to try and determine the chemical compounds responsible, and though there is still no definitive verdict as to the manner in which these compounds are formed, it has been suggested that they all form from asparagusic acid.

Asparagusic acid is, unsurprisingly considering the name, a chemical found exclusively in asparagus, and absent in other related vegetables.

The asparagus-pee molecules that you smell come mostly from the breakdown of a molecule known as asparagusic acid, which is present naturally in asparagus. When your body breaks down asparagusic acid it forms a wide variety of chemicals, all of which contain sulfur!

This has made it an obvious candidate for being the origin of the peculiar effect that asparagus has on urine. It has been suggested by recent studies that it could be metabolised in the body to produce the volatile compounds found in the urine after consuming the vegetable.

Asparagus has been used as a vegetable and medicine, owing to its delicate flavour, diuretic properties, and more. It is pictured as an offering on an Egyptian frieze dating to 3000 BC. Still in ancient times, it was known in Syria and in Spain. Greeks and Romans ate it fresh when in season and dried the vegetable for use in winter; Romans would even freeze it high in the Alps, for the Feast of Epicurus. Emperor Augustus tossed off the “Asparagus Fleet” for hauling the vegetable, and coined the expression “faster than cooking asparagus” for quick action. A recipefor cooking asparagus is in the oldest surviving book of recipes, Apicius’s third-century AD De re coquinaria, Book III.

The ancient Greek physician Galen (prominent among the Romans) mentioned asparagus as a beneficial herb during the second century AD, but after the Roman empire ended, asparagus drew little medieval attention. until al-Nafzawi‘s The Perfumed Garden. That piece of writing celebrates its (scientifically unconfirmed) aphrodisiacal power, a supposed virtue that the IndianAnanga Ranga attributes to “special phosphorus elements” that also counteract fatigue. By 1469, asparagus was cultivated in French monasteries. Asparagus appears to have been hardly noticed in England until 1538, and in Germany until 1542.

The finest texture and the strongest and yet most delicate taste is in the tips. The points d’amour (“love tips”) were served as a delicacy to Madame de Pompadour. Asparagus became available to the New World around 1850, in the United States.

German botanical illustration of asparagus

Chemistry

Asparagus foliage turns bright yellow in autumn

Certain compounds in asparagus are metabolized to yield ammonia and various sulfur-containing degradation products, including various thiols andthioesters, which give urine a characteristic smell.

Some of the volatile organic compounds responsible for the smell are:

• methanethiol
• dimethyl sulfide
• dimethyl disulfide
• bis(methylthio)methane
• dimethyl sulfoxide
• dimethyl sulfone

Subjectively, the first two are the most pungent, while the last two (sulfur-oxidized) give a sweet aroma. A mixture of these compounds form a “reconstituted asparagus urine” odor. This was first investigated in 1891 by Marceli Nencki, who attributed the smell to methanethiol. These compounds originate in the asparagus as asparagusic acid and its derivatives, as these are the only sulfur-containing compounds unique to asparagus. As these are more present in young asparagus, this accords with the observation that the smell is more pronounced after eating young asparagus. The biological mechanism for the production of these compounds is less clear.

The onset of the asparagus urine smell is remarkably rapid. The smell has been reported to be detectable 15 to 30 minutes after ingestion.

Gas chromatography-mass spectrometry was used to analyse the ‘headspace’ of urine produced after consumption of asparagus. The headspace is the gas space immediately above the liquid surface, which is occupied by light, volatile compounds in the liquid, and analysis of this is useful in identifying odour-causing compounds. The analysis of the post-asparagus urine showed the presence of several compounds that were not present, or present in negligible amounts, in normal urine. The primary compounds present, in quantities a thousand times greater than in normal urine, were methanethiol and dimethyl sulfide. The compounds dimethyl sulfide and dimethyl sulfone were also present and it was suggested that they modify the aroma to give it a ‘sweet’ edge.