WO2007112473A1

WO2007112473A1 - Purification of a difluoronucleoside anomer using aqueous acid solution

Info

Publication number: WO2007112473A1
Application number: PCT/AU2006/000430
Authority: WO
Inventors: Huijan Zhong; Aifeng Lv; Raju Kalidindi; Song Lin; Xiping Su
Original assignee: Mayne Pharma Limited; Chemwerth Inc; Jiangsu Hansen Pharmaceutical Co Ltd
Priority date: 2006-03-31
Filing date: 2006-03-31
Publication date: 2007-10-11

Abstract

A process for purifying a crude α- or a crude β-anomer of a 2,2'-difluoronucleoside, or a derivative thereof, which process includes: providing: an organic or inorganic acid; an amount of crude α- or β-anomer of a 2,2'-difluoronucleoside or a derivative thereof; and water; forming an aqueous solution of the crude α- or crude β-anomer in the water; adding a sufficient amount of the acid to the aqueous solution such that the pH of the resulting solution does not exceed a predetermined value; collecting a precipitate; and optionally subjecting the precipitate to one or more washing steps.

Description

PURIFICATION OF A DIFLUORONUCLEOSIDE ANOMER USING AQUEOUS ACID SOLUTION

Field of the invention

The present invention relates to a process for the selective isolation of a 2,2'- difluoronucleoside as a free base, or as an organic or inorganic addition salt, from a mixture of a- and β- anomers of the free base nucleoside.

Background of the invention

2,2'-Difluoronucleosides are known anti-viral and anti-tumour agents (US 4,808,614 and US 5,464,826). In particular, 1-(2-oxo-4-amino-1 H-pyrimidin-1-yl)-2-desoxy-2,2'- difluororibose (commonly known as Gemcitabine), has been shown to have anti-viral and anti-tumour activity. It has been found that the /?~anomer of Gemcitabine is the most potent, therefore it is desirable to have a synthetic process that results in simple and relatively high yielding isolation of the β-anomer of Gemcitabine.

Known processes for synthesising 2,2'-difluoronucleosides are generally multi-step processes, (for example, Gemcitabine, US 4,526,988) and the intermediate chiral compounds that are formed during the process are present as a mixture of anomers. It is not until near the end of the process that any attempt is made to isolate one of the anomers. In the case of Gemcitabine, the active /?-anomer is isolated from an approximately 4:1 mixture of a- and /?-Gemcitabine, meaning that the desired β- Gemcitabine is present as the minor component. The /?-anomer is separated using column chromatography, which is an expensive and arduous process.

US 5,223,608 describes a process for the generation of an anomeric mixture of Gemcitabine (ie, a:β mixture of Gemcitabine) in an about 1 :1 ratio, and the selective isolation of the /?-anomer from that mixture, either as the free base, or as an organic or inorganic acid addition salt. The /?-anomer may be selectively isolated as its hydrochloride or hydrobromide salt (in about 80.0% purity) by dissolving the 1:1 cr.β anomeric mixture in hot water, adding acetone, cooling the solution to between about -10⁰C and about 50⁰C and collecting the precipitated salt. To obtain the Gemcitabine hydrochloride or hydrobromide in a level of purity suitable for pharmaceutical applications, the product from this recrystallisation is subjected to the above process again.

US 5,223,608 also describes (in Example 8) the selective isolation of the hydrochloride salt of the β-anomer (in about 90% purity) from an approximately 1 :1 cr.β anomeric mixture of gemcitabine, by dissolving the anomeric mixture in concentrated hydrochloric acid (-38% HCI in water) and isopropanol, cooling the solution and collecting the precipitate. Example 10 of US 5,223,608 describes the selective isolation of the hydrobromide salt of the /?-anomer by a similar process to that described in Example 8.

In addition, US 5,223,608 describes the selective isolation of free base from an about 1:1 a:β anomeric mixture of the free base or of the organic or inorganic addition salt thereof by dissolving the cr.β anomeric mixture in hot water, increasing the pH of the resulting solution to between about 7.0 and 9.0, cooling the solution to a temperature between about -1O⁰C and about 30⁰C and collecting the resulting precipitate of Gemcitabine as its free base. This free base can subsequently be converted into an organic or inorganic addition salt if required.

It is, accordingly, an object of the present invention to overcome, or at least alleviate, one or more of the difficulties and deficiencies related to the prior art.

Summary of the invention In a first aspect of the present invention there is provided a process for purifying a crude a- or a crude /?-anomer of a 2,2'-difluoronucleoside, or a derivative thereof, which process includes:

providing:

an organic or inorganic acid; an amount of crude a- or β- anomer of a 2,2'-difluoronucleoside or a derivative thereof; and

water;

forming a solution of the crude a- or/?- anomer in the water;

adding a sufficient amount of the acid to the aqueous solution such that the pH of the resulting solution does not exceed a predetermined value;

collecting a precipitate; and

optionally subjecting the precipitate to one or more washing steps.

Detailed description of the embodiments As used herein in the specification and claims, the term "comprises" (or its grammatical variants) is equivalent to the term "includes" and should not be taken as excluding the presence of other elements or features.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission or any form of suggestion that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed at before the priority date of each claim of the application.

The term "crude σ-anomer" or "crude /?-anomer", as used herein in the specification and claims, refers to the fact that the materia! may not be pure a- or /?-anomer at that point.

While the crude σ-anomer or the crude ^-anomer may contain the σ-anomer or β- anomer, respectively, as the major component, various other compounds may be present in the material as impurities, including, for example, other anomers, enantiomers, salts, intermediates, starting materials, minor reaction products, and the like. The derivative may be of any suitable type known to the person skilled in the art. For example, the derivative may be a salt. An organic or inorganic addition salt is preferred.

The organic acid may be selected from the group consisting of but not limited to tartaric acid, citric acid, acetic acid, or salts thereof. The inorganic acid, may be selected from a halogenic acid, such as hydrochloric acid or hydrobromic acid, sulfuric acid, phosphoric acid, or salts thereof. Preferably, the acid is a halogenic acid, such as hydrochloric acid or hydrobromic acid. Most preferably, the acid is hydrochloric acid.

Preferably, the predetermined value that the pH of the resulting solution does not exceed is about 4.0, more preferably 2.0, most preferably 0.5.

Preferably the precipitate is washed with an alcohol. Where an alcohol is used, preferably the alcohol is isopropanol.

The applicants have surprisingly discovered that adjusting the pH of the solution to below about 4.0, preferably about 0.5 assists in the purification of the crude a- or crude /?-anomer. Without wishing to be bound by theory, we postulate that adjusting the pH of the solution accelerates the nucleation process during crystallisation, which may result in a more consistent and controlled crystallisation process, an increase in the speed of crystallisation and good quality crystals. It is also postulated that the yield from the crystallisation process is also assisted by the low solubility of the β-anomer in an acidic environment.

According to one preferred embodiment, the solution of the a- or /?-anomer is obtained by adding the a- or /?-anomer to water that has been heated to an elevated temperature. Preferably the water is heated to between about 40⁰C and about 70⁰C and more preferably between about 45⁰C and about 60⁰C.

The process of the present invention may further include the step of filtering the solution of the a- or /?-anomer prior to the addition of the acid. Optionally, the process further includes the step of cooling the solution after the addition of the acid. Preferably, the temperature to which the mixture of 2,2'-difluoronucleoside and water is cooled is between about -3°C and about 5°C. More preferably, the temperature is between about 0⁰C and about 4⁰C.

The process may additionally involve the step of further readjusting the pH of the solution after the cooling step, such that the pH does not exceed the predetermined value.

Preferably, the 2,2'-difluoronucleoside is 1-(2-oxo-4-amino-1 H-pyrimidin-1-yl)-2-desoxy- 2,2'-difluororibose.

The crude a- or crude /?-anomer of a 2,2'-difluoronucleoside or derivative thereof, may be produced by any suitable method. According to one preferred embodiment, the crude a- or crude /?-anomer is isolated from an aqueous solution of a mixture of a- and /?-anomers of the 2,2'-difluoronucleoside, or a derivative thereof, by adding an alcohol to the aqueous solution, collecting the resulting precipitate and optionally subjecting the precipitate to one or more washing steps.

According to one preferred embodiment of the present invention, the aqueous solvent is water.

Dissolution of the a- and β- anomers of the 2,2'-difluoronucleoside, or derivative thereof, in an aqueous solvent may be performed by any suitable means. For example, dissolution may occur with the assistance of stirring or heating of the mixture. In one preferred embodiment, the solution is both heated and stirred. According to one preferred embodiment, the aqueous solvent may be heated before the mixture of a- and β- anomers of the 2,2'-difluoronucleoside is added. When the aqueous solvent is heated, it may be heated to any suitable temperature, but preferably it is heated to between about 40⁰C and about 70⁰C, more preferably, between about 45°C and about Preferably, the alcohol is a short chain alcohol. More preferably the alcohol is a Ci_-4 alcohol, for example methanol, ethanol, n-propanol, isopropanol, n-butanol, f-butanol, and the like.

The optional washing step may be performed using any suitable solvent. According to one preferred embodiment, the solvent is an alcohol, preferably isopropanol. According to another preferred embodiment, the alcohol added to the aqueous solution is the same alcohol used in the optional washing of the precipitate.

The selective isolation process may include the further step of filtering the aqueous solution prior to the addition of the alcohol to the filtrate produced.

Optionally, the selective isolation process further includes the step of cooling the aqueous solution or allowing the aqueous solution to cool after the addition of the alcohol. The aqueous solution may be cooled to between about 5⁰C and about 35°C, more preferably between about 1O⁰C and about 30⁰C, most preferably between about 15°C and about 25⁰C.

Preferably the precipitate is collected by filtration.

Preferably, the 2,2'-difluoronucleoside is 1-(2-oxo-4-amino-1 H-pyrimidin-1-yl)-2-desoxy- 2,2'-difluororibose (Gemcitabine). The mixture of a- and β- anomers of Gemcitabine may be prepared by processes well known to those skilled in the art.

Preferably, the /?-anomer is selectively isolated.

In another aspect of the present invention there is provided a process for the production of an organic or inorganic acid addition salt of a 2,2'-difluoronucleoside. According to this aspect, the free base is used as a starting material and the pH adjustment has the added effect of forming the acid addition salt of the 2,2'-difluoronucleoside.

The process includes: providing

a free base 2,2'-difluoronucleoside

an organic or inorganic acid; and

water;

heating the water to an elevated temperature;

dissolving the free base 2,2'-difluoronucleoside in water;

adding an organic or inorganic acid to the solution in an amount sufficient to adjust the pH of the resulting solution to a predetermined value;

cooling the resulting solution;

collecting a precipitate, and

optionally subjecting the precipitate to one or more washing steps.

In a further aspect of the present invention there is provided an a- or a β-2,7!- difluoronucleoside, or a derivative thereof prepared by a process as described above.

In a still further aspect of the present invention there is provided an organic or inorganic acid addition salt of a 2,2'-dif!uoronucleoside prepared from the free base 2,2- difluoronucleoside as described above.

As stated above, the 2'-deoxy-2',2'-difluoronuc!eosides are useful antiviral or oncolytic agents. The active component may be included in a pharmaceutical composition. The pharmaceutical composition may include standard ingredients, including suitable solvents, fillers, colourants and the like. The active component may be used alone, or in combination with other actives. The present invention will now be more fully described with reference to the accompanying examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

Brief description of the drawings

In the drawings:

Figure 1 is a reaction sequence in relation to a process according to one preferred embodiment of the present invention.

Figure 2 is a flow-chart depicting sequential steps in a resolution process according to the preferred embodiment of the invention described in Figure 1.

Figure 3 is a flow-chart depicting sequential steps in a purification process according to the invention.

EXAMPLES

Resolution ofi5-1-(2-oxo-4-amino-1H-pyrimidin-1-yl)-2-desoxy-2,2'-difluororibose hydrochloride from βr//?-1-(2-oxo-4-amino-1 H-pyrimidin-1-yl)-2-desoxy-2,2'- difluororibose hydrochloride

Purified water (25 L) was discharged into a 500 L reactor and heated to 55±5 ⁰C for 30 minutes. The reactor was charged with σ//M-(2-oxo-4-amino-1H-pyrimidin-1-yl)-2- desoxy-2,2 -difluororibose hydrochloride (3.57 kg) and the mixture was stirred to ensure dissolution. The mixture was then filtered, lsopropanol (250 L) was added to the filtrate. The mixture was stirred for 2 hours and allowed to cool to about 25°C.

The solution was then filtered and the solid washed. The precipitate was then placed on a tray and covered with a cloth. The tray was placed into a vacuum dryer for at least 6 hours at a temperature of 40±5 ⁰C at a vacuum of about <0.07 MPa resulting in /?-1- (2-oxo-4-amino-1H-pyrimidin-1-yl)-2-desoxy-2,2^f-difluororibose hydrochloride (1 b) being obtained as-a white powder (1.035 kg, 98% purity as determined by HPLC).

Preparation of/M-(2-oxo-4-amino-1H-pyrimidin-1-yl)-2-desoxy-2,2'-difluororibose hydrochloride

Purified water (42 L) was discharged into a 100 L reactor and heated to 55±5°C. The reactor is then charged with crude /M-(2-oxo-4-amino-1 H-pyrimidin-1-yl)-2-desoxy-2,2'- difluororibose hydrochloride (4.14 kg) prepared according to the above example. The solution was then stirred for approximately 30 minutes and then filtered using a Buchner funnel. The pH of the filtrate was adjusted to <0.5 with concentrated hydrochloric acid (approximately 500 mL). The solution was stirred and cooled to about 0⁰C for 1 hour.

The resulting precipitate was collected on a 250 mm Buchner funnel, and washed with isopropanol (3 x 3 L). The precipitate was then placed on a tray and covered with a cloth. The tray was placed into a vacuum oven for at least 6 hours at a temperature of 40±5 ⁰C at a vacuum of about <0.07 MPa resulting in /?-1-(2-oxo-4-amino-1 H-pyrimidin- 1-y!)-2-desoxy-2,2'-difluororibose hydrochloride being obtained as a white powder (3.6 kg, 99.8% purity as determined by HPLC).

Claims

1. A process for purifying a crude a- or a crude /?-anomer of a 2,2'- difluoronucleoside, or a derivative thereof, which process includes

providing:

an organic or inorganic acid;

an amount of crude a- or /?-anomer of a 2,2'-difluoronucleoside or a derivative thereof; and

water;

forming an aqueous solution of the crude a- or crude /?-anomer in the water;

collecting a precipitate; and

optionally subjecting the precipitate to one or more washing steps.

2. A process according to claim 1 wherein the pH of the resulting solution does not exceed about 4.0.

3. A process according to claim 2 wherein the pH of the resulting solution does not exceed about 2.0.

4. A process according to claim 3 wherein the pH of the resulting solution does not exceed about 0.5.

5. A process according to claim 1 wherein the acid is a halogenic acid.

6. A process according to claim 5 wherein the acid is hydrochloric acid or hydrobromic acid.

7. A process according to claimi wherein the derivative of the 2,2'-difluronucleoside is an organic or inorganic addition salt.

8. A process according to claim 1 wherein the precipitate is washed with an alcohol.

9. A process according to claim 8 wherein the alcohol is isopropanol.

10. A process according to claim 22 wherein the aqueous solution of the crude a- or crude /?-anomer is obtained by adding the crude a- or crude /?-anomer to water heated to an elevated temperature.

11. A process according to claim 10 wherein the water is heated to between about 45⁰C and 65⁰C.

12. A process according to claim 1 further including the step of filtering the aqueous solution of the crude a- or crude /?-anomer prior to the addition of the acid.

13. A process according to claim 1 further including the step of cooling the resulting solution after the addition of the acid.

14. A process according to claim 13 wherein the resulting solution is cooled to between about -3°C and about 5°C.

15. A process according to claim 13 further including the step of readjusting the pH after the cooling step such that the pH of the solution does not exceed the predetermined value.

16. A process according to claim 15 wherein the pH does not exceed about 4.0.

17. A process according to claim 16 wherein the pH does not exceed about 2.0.

18. A process according to claim 17 wherein the pH does not exceed about 0.5.

19. A process according to claim 1 wherein the 2,2'-difiuoronucleoside is 1-(2-oxo-4- amino-1 H-pyrimidin-1 -yl)-2-desoxy-2,2'-difluororibose.

20. A process according to claim 19 wherein the crude /?-anomer of 1-(2-oxo-4~ amino-1 H-pyrimidin-1 -yl)-2-desoxy-2,2'-difluororibose is purified.

21. A process according to claim 1 wherein the crude a- or crude /?-anomer is obtained by a process including:

providing

a mixture of a- and /?-anomers of a 2,2'-difluoronucleoside or a derivative thereof; and

an aqueous solvent;

dissolving the mixture of a- and /?-anomers in the aqueous solvent;

adding an alcohol to the resulting aqueous solution;

collecting a precipitate; and

optionally subjecting the precipitate to one or more washing steps.

22. A process for the production of an organic or inorganic acid addition salt of a 2,2'- difluoronucleoside, which process includes:

providing

a free base 2,2'-difluoronucleoside;

an organic or inorganic acid; and water

heating the water to an elevated temperature;

dissolving the free base 2,2'-difluoronucleoside in the water;

cooling the resulting solution;

collecting a precipitate; and

optionally subjecting the precipitate to one or more washing steps.

23. A process according to claim 22 wherein the pH of the resulting solution does not exceed about 4.0.

24. A process according to claim 23 wherein the pH of the resulting solution does not exceed about 2.0.

25. A process according to claim 24 wherein the pH of the resulting solution does not exceed about 0.5.

26. A process according to claim 22 wherein the acid is a halogenic acid.

27. A process according to claim 26 wherein the acid is hydrochloric acid or hydrobromic acid.

28. A process according to claim 22 wherein the pH adjusted solution is cooled to a temperature between about -2°C and about 3°C.

29. A process according to claim 22 wherein the precipitate is washed with an alcohol.

30. A process according to claim 22 further including the step of readjusting the pH of after the cooling step such that the pH of the solution does not exceed the predetermined value.

31. A process according to claim 22 wherein the 2,2'-difluoronucleoside is 1-(2-oxo- 4-amino-1 H-pyrimidin-1-yl)-2-desoxy-2,2'-difluororibose.

32. A process according to claim 22 wherein the water is heated to between about 45°C and 65°C.

33. An a- or a /?-2,2'difluoronucleoside, or a derivative thereof, prepared by a process according to claim 1.

34. An a- or a /?-2,2'-difluoronucleoside according to claim 33 wherein the derivative is an organic or inorganic acid addition salt.

35. A organic or inorganic addition salt of a 2,2'-difluoronucleoside prepared from the free base 2,2'-difluoronucleoside according to a process according to claim 22.

36. A pharmaceutical composition including an a- or a /?-2,2'-difluoronucleoside, or a derivative thereof according to claim 33.

37. A pharmaceutical composition including an organic or inorganic acid addition salt of a 2,2'-difluoronucleoside according to claim 34.

38. A pharmaceutical composition including an organic or inorganic acid addition salt of a 2,2'-difluoronucleoside according to claim 35.