WO2016097989A1

WO2016097989A1 - Process for the preparation of gemcitabine hydrochloride

Info

Publication number: WO2016097989A1
Application number: PCT/IB2015/059620
Authority: WO
Inventors: Khashayar Karimian; Ahmad YARI
Original assignee: Khashayar Karimian
Priority date: 2014-12-16
Filing date: 2015-12-15
Publication date: 2016-06-23

Abstract

The present invention relates to an improved as well as an industrially viable process for the preparation of 2'-deoxy-2',2'-difluorocytidine and its pharmaceutical acceptable acid salts thereof in high purity and acceptable yield by using a simple and inexpensive process. The process involves reacting an alpha anomer enriched 2'-deoxy-2',2'-difluorocarbohydrate with silylated nucleobase derivatives via the SN2 displacement of an anomeric sulfonyloxy group in an inert solvent to produce 2',2'-difluoro-2'-deoxycytidine-3',5'-dibenzoate in about a 5:1 β/α anomeric ratio, and a process for selectively isolating β-2',2'-difluoro-2'-deoxycytidine-3',5'- dibenzoate from the 5:1 β/α anomeric mixture. The pure β-2',2'-difluoro-2'- deoxycytidine-3',5'-dibenzoate is then converted to the corresponding nucleoside utilizing ammonium hydroxide in a polar and preferably protic solvent to obtain β- 2'-deoxy-2',2'-difluorocytidine, which is thereafter converted to gemcitabine hydrochloride that is effective against non-small cell lung cancer, pancreatic cancer, bladder cancer and breast cancer. The present invention relates to an improved and convenient method for preparing antineoplastic nucleosides, more particularly, a process for preparing gemcitabine hydrochloride, represented by the formula I below, which exhibits good antitumor activity. (I)

Description

PROCESS FOR THE PREPARATION OF GEMCITABINE HYDROCHLORIDE

FIELD OF THE INVENTION

The present invention relates to an improved as well as an industrially viable process for the preparation of 2'-deoxy-2',2'-difluorocytidine and its pharmaceutical acceptable acid salts thereof in high purity and acceptable yield by using a simple and inexpensive process.

BACKGROUND OF THE INVENTION

It has been known that 2'-deoxynucleosides and their analogues are useful antiviral agents. These compounds exhibit superior activity in the treatment of various cancers. Among them, 2'-deoxy-2',2'-difluorocytidine of formula II which is generically known as gemcitabine and chemically described as 4-amino-1 -(2- deoxy-2,2-difluoro-p-D-ribofuranosyl)pyrimidin-2(1 H)-on, belongs to a group of chemotherapy drugs known as antimetabolites. Gemcitabine is a pyrimidine which has a cytosine nucleobase at the C position of the ribofuranose and stereochemical ly oriented to β-direction and has been shown to be effective for the treatment of many types of cancers including: non-small cell lung cancer, pancreatic cancer, bladder cancer and breast cancer. Gemcitabine is represented by the following structure:

(Π)

There have heretofore been many processes for the synthesis difluoronucleosides in the art. Process for preparing 2'-deoxy-2',2'-difluorocytidi involves reacting of an activated ribofuranose having a reactive leaving group at the anomeric centre, as shown in the reaction scheme 1 , with a silylated nucleobase such as cytosine and its derivatives wherein the leaving group at the anomeric carbon is replaced with the nucleobase.

L (reactive leaving group): Sulfonate, halide or imidate. ^

R_[ and R₂: Benzoyl, phenylbenzoyl, TBDPhS, TBDMS, cinnamoyl, trityl, phenylcarbamoyl or naphtoyl.

R_[ and R₂ are the same or different.

While not wishing to be bound by theory, it is believed that the glycosylation reaction proceeds primarily via S_N2 displacement. Therefore, when a β-anomer nucleoside is desired, an a-anomer enriched carbohydrate is preferably used in SN2 coupling reaction to decrease the amount of undesired a-anomer nucleoside product. The critical and cumbersome step in the preparation of the aforesaid nucleoside is the step of N-glycoside bond formation wherein the leaving group of the carbohydrate is replaced with the protected nucleobase. The process for preparing 2'-deoxynucleosides is typically non-stereoselective and a mixture of alpha and beta anomer is obtained. It must be considered even when the leaving group at the anomeric center has alpha stereochemistry, mixture of isomers are formed. It is believed and also our experiments showed that when the pure 1 - alpha ribofuranose methanesulfonate is reacted with trimethylsilylmethanesulfonate in an inert solvent such as toluene and xyelene at reflux temperature, trimethylsilylmethanesulfonate anomerized the alpha ribofuranose and a mixture of alpha and beta carbohydrate is obtained and as a result, a mixture of nucleosides is produced in the reaction condition.

The known processes for preparing difluoronucleosides are summarized herein below. Hertal et al firstly disclosed the 2'-deoxy-2',2'-difluorocytidine compound in the Eur. Pat. No. 0,184,365 A2, J. Org. Chem. 53, 2406 (1988), US. Pat. Nos. 5.269.988 and 4,808,614 and described the use of the titled compound as antiviral agent as well as their preparation. The ribofuranosyl derivative of 3,5-bis(t-butyldimethyl silyloxy)-1 -methanesulfonyloxy-2'-deoxy-2',2'-difluororibose is prepared in five steps and thereafter the reaction followed by condensing the abovementioned carbohydrate with silylated N-acetylcytosine in the precense of trimethylsilyl trifluoromethanesulfonate (TMSOTf) as Lewis acid and reaction initiator. After the reaction is complete, the product composed of about 4: 1 α /β anomeric ratio. The applicant utilized several different stages as well as a large quantity of solvents to isolate the mixture of alpha and beta anomers. To obtain pure p-2'-deoxy-2',2'- difluorocytidine, column chromatography is used, resulting in low yield which is not suitable for large scale industrial application. Also, the separation requires expensive silica gel as column chromatography and large volumes of solvents, affording an industrially unviable process.

U.S. Pat. Nos. 4,965,374 and 5.223.608 teaches a process for the preparation of gemcitabine which discloses utilizing a new ribofuranosyl in which C3 and C5 are both protected by benzoyl protecting groups. The process comprises reacting 2'- deoxy-2',2'-difluoro-D-erythro-pentofuranose-3,5-dibenzo ate-1 -methanesulfonate with bis-(trimethylsilyl)-N-acetylcytosine in the precense of trimethylsilyl triflate as reaction initiator. The difluoronucleoside is obtained as the dibenzoate ester and the product is as a mixture of beta and alpha anomers with about 1 :1 anomeric ratio. After the esters and silyl protecting groups are removed from the anomeric mixture, a and p-2-deoxy-2,2-difluorocytidine are obtaind in about 1 :1 anomeric ratio with the yield of 47% (both isomers). The patent describes isolating the β-2- deoxy-2,2-difluorocytidine by dissolving 1 :1 anomeric mixture of -2-deoxy-2,2- difluorocytidine in hot water, raising the pH to 7-9, cooling to a temperature of about -10-30°C and finally collecting the precipitated p-2'-deoxy-2',2'- difluorocytidine free base. The p-2'-deoxy-2',2'-difluorocytidine is then converted to its hydrochloride salt by dissolving the free base in hot isopropanol, followed by addition of concentrated HCI. Despite the advantages of the aforesaid process compared to the prior art and keeping in mind that the invention eliminates the need for expensive column chromatography purification, the process is non- selective affording a 1 :1 anomeric mixture in low yield. Again, this does not constitute an industrially viable process. In addition, the methods disclosed in abovementioned patents, suffer from the disadvantage in that repeated crystallization steps are required to obtain the product of 99% purity, not only increasing the length but also the cost of manufacture.

U.S. Pat. 5,521 ,297 utilizes a new ribofuranosyl intermediate wherein 3-hydroxy group in the carbohydrate is protected by different carbamoyl derivatives to produce difluoronucleoside. The patent claims using 3-hydroxy carbamoyl group on the difluororibose intermediate enhances formation of the desired beta anomer nucleoside derivative by favoring attack by the silylated nucleobase from the opposite side and as a result, beta-anomer nucleoside derivative is preferably formed. In an embodiment of the invention, 3-carbamoyl-5-benzoyl-2'-deoxy-2',2'- difluororibose-1 -alpha-mesylate is reacted with silylated cytosine in xylene in the presence of trimethylsilyl triflate. After the removal of protecting group by sodium methoxide, the product is composed of about 60:40 β/α anomeric mixture with in situ HPLC purity of 59%. Although the improvement of the process compared to those used the same protecting groups at the C3 and C5 of the carbohydrate (see U.S. Pat. No. 4.965.374) and increasing the β la anomeric ratio from 50:50 to 60:40, the process still suffers from non-selectivity as well as using an explosive, corrosive and expensive Lewis acid which makes the process less favored for large scale production.

Further, there have been reported haloribofuranosyl derivative intermediates for preparing difluoronucleosides. For example, U.S. Pat. No. 8,193,339 B2 describes a process for preparing gemcitabine wherein 4,4-difluoro-5-iodo-2- (trityloxymethyl)tetrahydrofuran-3-yl-benzoate is reacted with silylated cytosine in the presence of silver carbonate in acetonitrile. The protected difluoro nucleoside product is composed of both beta and alpha anomers with about 5.6:1 β/α anomeric ratio with quantitative yield. In the next step, after the removal of the trityl protecting group, beta and alpha hydrochloride salt is obtained with about 28% yield. Thereafter, the anomeric mixture of titled nucleoside was treated with ammonia/methanol to obtain gemcitabine free base with the yield of about 85%. In spite of the novelty of the foresaid process and utilizing new carbohydrate derivative, the low overall yield (24%) of the process makes it difficult to be industrially feasible.

U.S. Pat. 7,799,907 also teaches the production of gemcitabine. Specifically, 1 -a- bromo-2'-deoxy-2',2'-difluoro -D-ribofuranosyl-5-benzoyl-3-(4-phenyl)benzoate is reacted with at least 15 molar equivalents of silyl-protected cytosine in a mixture of octane and diphenylether at the elevated temperature of about 140-150°C. The process involves continuous removal of released trimethylsilyl bromide by unremitting addition of diphenylether/heptane mixture to avoid the anomerization of furanosyl bromide. The β/α anomeric ratio rages from 4.9:1 to 14:1 depending on the nucleobase/furanosyl molar ratio, which varies from 15 to 41 molar equivalents. The pure p-2'-deoxy-2',2'-difluorocytidine is obtained in an overall yield of 72.6% which makes the process industrially attractive, but it still suffers from the large quantity of the solvents used in the process which need then to be recovered from in relatively pure form while the solvents are immiscible with water and have close boiling point .

Other known intermediates used in the synthesis of gemcitabine include, t- butyldiphenylated intermediate (U.S. Pub. No. 2009/0069557 A1 and 2007/015257 A2), naphtoylated intermediate (U.S. Pub. No. 2010/0069625 A1 ), cinnamoylated intermediate (WO 2008/129530 A1 ) and tritylated intermediate (U.S. Pat. No. 5,559,22).

A catalytic glycosylation process for preparing difluoronucleosides has also been reported. For example, U.S. Pat. No. 5,426,183 describes a catalytic stereoselective process for preparation of 2'-deoxy-2',2'-D-ribofuranosyl difluoronucleosides. Specifically, 2'-deoxy-2',2'-difluoro-D-erythro-pentofuranose- 3,5-dib enzoate-1 -a-methanesulfonate is reacted with an excess amount of nucleobase derivative in a molar ratio ranging from about 10 molar equivalents to 12.6 molar equivalents to produce a mixture of a and β anomers in a β/α anomeric ratio of ranging from 3:1 to 58.4:1 depending on the nature of the catalyst used. The catalysts of the process selected from the group comprising potassium, barium, cesium and trialkyl salts of trifluoromethanesulfonic acid, nanofluorobutanesulfonic acid etc. The applicant does not report any isolation/purification method as well as the isolated yield. Anionic glycosylation process for preparing 2'-deoxy-2',2'-difluoronucleosides is also known. For example, U.S. Pat. No. 5,744,597 discloses the use of anionic nucleobase derivatives for preparing such nucleosides. The process involves reacting N-pivaoyl cytosine with potassium t-butoxide to obtain potassium salt of N-pivaoyl cytosine. Thereafter, 2'-deoxy-2',2'-difluoro-D-ribofuranosyl-3,5- dibenzoyl-1 -a-(p-bromobenze ne)sulfonate is reacted with potassium salt of N- pivaoyl cytosine in acetonitrile to form a and β anomers in about 3.9: 1 β/α anomeric ratio. To isolate the beta-enriched anomer, column chromatography is used to obtain the p-N⁴-pivanoyl-2'-deoxy-2',2'-difluorocytidine with the yield limitied to 20%. Meanwhile, the patent describes when 2'-deoxy-2',2'-difluoro-D- ribofuranosyl-3,5-dibenzoyl-1 -a-iodide is used as carbohydrate source in the same reaction condition as described above, a and β anomers in about 1 .13:1 β/α anomeric ratio are obtained. Utilizing column chromatography to isolate the product as well as the low yield of the process are the shortcomings of the process which renders it not at all attractive on a commercial scale.

Ribofuranosyl derivatives of imidates as leaving group at the anomeric centre have been reported for preparing difluoro nucleosides. For example, U.S. Pat. 8,168,766 rreports a process for producing 2'-deoxy-2',2'-difluoro nusleosides wherein the carbohydrate derivatives is activated by various imidates such as, Ν,Ν-dicyclohexylcarbamimidate, N,N-dicyclohexyl-N-mesyl-carbamimidate, 1 ,1 ,3,3-tetramethyl-isouronium triflate and 1 ,1 ,3,3-tetramethyl-isouronium chloride at the 1 -position of the ribofuranosyl derivatives. The process comprises reacting the abovementioned imidate-activated carbohydrate with about 20-30 molar equivalents of the silylated cytosine to form a mixture of beta and alpha anomers. U.S. Pat. 8,168,76 does give any details on beta and alpha anomeric ratio as well as removal of O-protecting group. However, the novelty of the process in using an activated carbohydrate does not afford sufficient justification for its applicability to industrial scale production of gemcitabine. The process uses expensive, hazardous and corrosive reagents such as oxalyl chloride, triflic anhydride and TMSOTf, which makes the invention difficult to apply for large scale production.

Furthermore, Eur. Pat. No. 1 ,853,616 B1 discloses a new ribofuranosyl derivative wherein the carbohydrate is activated by trichloroacetimidate as reactive leaving group at the 1 -position. Further, the production of difluoronucleosides is known. For example, U.S. Pat. No. 5,633,367 reports a process for producing 2'-deoxy-2',2'-difluorocytidine which involves reacting N⁴-3',5'-tri(O-toluoyl)-2'-ketocytidine with diethylaminosulfur trifluoride (DAST) and pyridine. HF in dichloromethane to yield 42% of N⁴-3',5'- tri(O-toluoyl)-2',2'-difluorocytidine, which is then converted to 2-deoxy-2,2- difluorocytidine utilizing sodium methoxide. In spite of the disclosure of a new intermediate for preparing difluoronucleosides, the aforementioned technique is deficient in that the product is obtained in low yield and also the process utilizes corrosive, flammable and expensive reagents.

Most of the aforementioned processes are cumbersome, complicated and expensive. The use of column chromatography renders them practically impossible to use on industrial scale and some are dangerous for application in large scale. Also, most reported processes require repeated crystallization to obtain pure gemcitabine. On the other hand, the prior art for isolating beta anomer involves the conversion of its hydrochloride to the free base followed by converting the obtained free base to the hydrochloride. Clearly, the said process increases the cost of the manufacture and reduces the overall yield of the product.

Furthermore, the prior art processes for the production of gemcitabine and its analogues are deficient in that the purified product is obtained by utilization expensive hydroxy protective group like tert-butyldimethylsilyl group and also applying Lewis acids and reagents including TMSOTf, Oxalyl chloride, DAST etc. which are hazardous, corrosive and expensive and therefore requiring special care and handling, thereby increasing the cost and risk of manufacture. Additionally, utilization of high boiling solvents and elevated reaction temperatures as well as the use of mixture of solvents for the preparation of gemcitabine hydrochloride result in high energy consumption.

Therefore, a need exists for an improved method for manufacture of 2'-deoxy-2',2'- D-ribofuranosyl difluoronucleosides which simplifies the isolation and purification process and also is selective for beta-2'-deoxy-2',2'-difluorocytidine giving acceptable yield and high purity and as a result, reduces not only the length but also the cost of the process. Moreover, there is a need of a simple and inexpensive process which is also industrially feasible for the isolation and purification of gemcitabine hydrochloride which eliminates the use of highly corrosive and hazardous Lewis acids and other reagents which do not require any special care and handling, thereby decreasing the cost and risk of manufacture.

It would be desirable to have a relatively simple process for producing gemcitabine in acceptable and/or comparable yields to reported methods. Furthermore, it would be desirable to have a simple process for the production of gemcitabine which is devoid of the limitations of the prior art.

BRIEF DESCRIPTION OF THE INVENTION

In the present invention, nucleobases of cytosine (or its derivatives) is reacted with a silyl protecting group shuch as hexamethyldisilazane (HMDS) in the presence of catalytical amount of ammonium sulfate in small portion of an inert organic solvent sush as toluene at a temperature of about 1 10-130 °C to produce the silylated nucleobase of formula (IX). Thereafter, the glycosylation reaction is conducted by the slow addition of a-methanesulfunate ribofuranosyl derivative solution of formula (X) to the silylated nucleobase mixture to obtaine the blocked 2'-deoxy- 2',2'-difluoronusleosides (XI) in about 5: 1 β/α anomeric ratio at the same temperature as mentioned above. After completing of the reaction, the reaction mixture is cooled to the ambient temperature and then a dilute solution of HCI was added thereto followed by filtration of the precipitate which contains hydrochloride salt of a and p-2'-deoxy-3',5'-dibenoate-2',2'-difluoronusleoside of formula (XII). Thereafter, the precipitate is taken in a polar and preferably protic organic solvent and heated to reflux followed by the addition of an alkyl amine and then cooled to room temperature and filtered to produce the pure p-2'-deoxy-3',5'-dibenoate- 2',2'-difluoronusleoside of formula (XIII). After removal of the hydroxyl protecting group of the carbohydrate utilizing aqueous ammonia and adjusting the pH to about 3, pure gemcitabine hydrochloride (I) is obtained with acceptable yield and high purity.

SUMMARY OF THE INVENTION

It is a primary object of the present invention to provide an efficient and simplified process for producing gemcitabine hydrochloride of the following formula under a new stereoselective glycolysation reaction using a pure a-methanesulfunate ribofuranosyl derivative of formula (V).

(I)

Another object of the present invention is to provide an efficient, convenient, cost effective and industrially feasible process for obtaining, in greater than 99.8% purity, nucleoside of the formula I.

It is a further object of the present invention to provide a process for glycolysation reaction to eliminate the use of Lewis acids such as TMSTOf and the difficulties associated with such reagents, which has been utilized in the prior art.

It is yet another object of the present invention to provide a process for selective isolation of p-2'-deoxy-3',5'-dibenoate-2',2'-difluorocytidine of the following formula V, comprising dissolving of a and p-2'-deoxy-3',5'-dibenoate-2',2'-difluorocutidine hydrochloride from the mixture of alpha and beta anomers in a polar and preferably protic solvent and selectively isolating it by using an alkyl amine.

(III) The present invention is described in more detail as set forth hereunder.

The process comprises protecting of cytosine and its derivatives with a silyl protecting group ant optionally in the presence of a catalyst in an inert non- nucleophilic organic solvent to obtain a silyl protected nucleobase of the following structure:

The bases set forth above are commonly known to organic chemists, and no discussion of their synthesis is necessary. However, the primary amino group, present on some of these bases should be protected before the nucleobase is coupled with the carbohydrate. The common amino-protecting groups include trimethylsilyl, t-buthyldimethylsilyl, benzyloxycarbonyl, formyl, acetyl and the like may be used according to the procedures known in the standard textbooks.

Coupling the nucleobase of the formula (IV) with the activated ribofuranosyl derivative of the following formula:

Ri

(V)

Wherein, R¹ is selected from the group consisting and not limited to benzoyl, nitrobenzyl, chloroacetyl, phenylbenzoyl, t-butyldiphenyl silyl, phenoxy acetyl, isobutyryl, ethoxy carbonyl, benzyloxy carbonyl, mesyl, trimethylsilyl, isopropyl dimethylsilyl, methyldiisopropyl silyl, triisopropyl silyl, t-butyldimethyl silyl, cinnamoyl, trityl, phenylcarbamoyl, phenoxycarbonyl, or naphtoyl, preferably, benzoyl. X is selected from the group comprising alkylsulfonyl, arylsulfonyl, substituted alkylsulfonyl, substituted arylsulfonyl, preferably, methanesulfonyl. R² is independentaly silyl protecting group and selected from the group consisting of C1-C7 trialkylsilyl, preferably, trimethylsilyl and R³ is H or acyl group to give the cytidine compound of formula (VI) which is composed of both alpha and beta anomers of the following structure:

Wherein R¹ , R² and R³ are the same as defined above.

Removal of the protecting groups R² and R³ of formula (VI) using dilute HCI to give the mixture of hydrochloride salt isomers of the compound of the formula (VII) as represented hereinbelow:

Wherein R¹ is the same as defined above.

Dissolving a compound of formula (VII) in a polar and preferably protic solvent/s followied by the addition of an alkyl amine to give the pure beta anomer of the following structure:

(VIII)

Wherein R¹ is the same as defined hereinbefore.

Deprotecting of the cytidine compound of formula (VIII) using deprotecting agent/s to give the p-2'-deoxy-2',2'-difluorocutidine of the formula (II):

Treating a compound of the formula (II) with concentrated halo acid to give gemcitabine halide of formula (I):

(I)

DETAILED DESCRIPTION OF THE INVENTION

The inventive method is characterized by limitation of the anomerization of the compound of formula (V), which can be converted to compound II in acceptable yield and in high purity. It is believed that the released trimethylsilylmethanesulfonate, which is produced during the glycosylation, anomerizes the ribifuranosyl compound and thus results in a mixure of anomers. Utilizing an excess amount of the nucleobase of formula (IV) results in high streoselectivity as well as increased β/α anomeric ratio.

The term "anomer-enriched' used herein means an anomer having a specific anomer content of greater

than 98%, including a substantially pure anomer. Also the term "anomerization" means that substantially pure anomer is epimerized at the Crposition of a ribofuranose.

Preferably, the process for producing a compound of Formula (II) can be used to produce various nucleosides such as, D-ribofuranosyl difluoronucleosides and derivatives, D-ribofuranosyl fluoronucleosides and derivatives, uridine and analogues, thymidine and analogues, cytidine and analouges or pharmaceutically acceptable salts thereof.

In accordance with the present invention, the streoselective glycolysation is carried out as shown in entire schematic representation: Preferably cytosine is treated with hexamethyldisilazane (HMDS) in the presence of a catalytic amount of (NH₄)₂SO₄ in a small portion of an inert solvent. Typically, the nucleobase protection reaction can be conducted at a temperature between 1 10 to 130°C and more preferably at 125°C. The nucleobase/(NH₄)₂SO₄ molar ratio is selected ranging from 15 to 30 molar equivalents and more preferably 20 molar equivalents. The HMDS/nucleobase molar ratio is selected ranging from 1 to 1 .2 molar equivalents and more preferably 1 .02 molar equivalents. e₃

The coupling reaction between the silylated base and carbohydrate may be carried out by any of the procedures known in the art. A preferred coupling procedure for preparing a compound of formula (XI) is comprised of reacting a compound of formula (IX) with a compound of formula (X) in an inert solvent at a temperature of about 1 10 to 130°C and more preferably at 125°C. Suitable inert organic solvents that can be employed are those which are water-immiscible. Therefore, apart from their non-participation in the essential reaction, such solvents form a two-phase system with water. Such a solvent offers advantage in not only effecting an efficient conversion but also helps in isolation of the product by simple evaporation or through work up by addition of water. Suitable inert water-immiscible organic solvents that can be employed include halogenated e.g. chlorinated hydrocarbons, e.g. dichloromethane, chloroform, tetrachloroethylene and 1 ,2-dichloroethane; esters e.g. (Ci_-4) alkyl esters e.g. ethyl acetate; ethers e.g. diisopropylether; aromatic hydrocarbons e.g. toluene, xylenes etc. aromatic hydrocarbons are preferred and amongst these toluene and xyelene are preferred and more preferably the suitable solvent for use is toluene.

Typically, the compound of formula (X) was dissolved in toluene and was slowly added to the protected nucleobase mixture within 6 hours. Preferably, the compound of formula (X) was dissolved in 6 w/v (nucleobase) of the solvent. The nucleobase/ribofuranosyl molar ratio is selected ranging from 10 to 20 molar equivalents and more preferably 15 molar equivalents. After completion of the reaction, the compound of formula (XI) is obtained in a β/α anomeric ratio of greater than 1 :1 and preferably greater than 3:1 and more preferably of about 5:1 β/α anomeric ratio.

The removal of silyl protecting group is achieved by using dilute halo acid, such as hydrochloric acid, followed by filtration of the precipitant to give a hydrodhoride salt of a compound of formula (XII) in about 5: 1 β/α anomeric ratio. The volume of the water used to precipitate the compound of formula (XII) ranges from about 3 to 4.5 w/v to the nucleobase and preferably is about 4 w/v. The HCI/nucleobase molar ratio used in this invention ranges from 1 .1 to 1 .5 molar equivalents and preferably 1 .3 molar equivalents. The removal of the silyl protecting group can be conducted at a temperature of 10 to 40°C and preferably at ambient temperature by the slow addition of water.

(XI) (XII) The process for preparing a compound of formula (XIII) is comprised of dissolving a compound of formula (XII) in a polar and preferably protic solvent at reflux temperature followed by the addition of an alkyl amine to selectively isolate the title compound. The solvent used herein to isolate the title compound is selected from the group comprising alcohols, ketones, esters, amides and nitriles. Preferably, the solvent is selected from the group comprising ethanol, isopropanol, methanol, ethyl acetate, acetone and more preferably methanol. The alkyl amine is selected from the group comprising C5-C12 hetrocyclic amines and amines having the general formula R⁴R⁵R⁶N wherein R^4, R⁵ and R⁶ can be the same or different and are selected from the group comprising hydrogen, a CrC₆ alkyl group and a C6-C9 aryl group, with the proviso that the each of R^4, R⁵ and R⁶ are not hydrogen. Thus, it will be appreciated that the use of ammonia (i. e. R4=R5=R6=H) is outside the scope of the present invention. Non-limiting examples of suitable hetrocyclic amines include pyridine and piperidine. Non- limiting examples of other amines suitable include t-butylamine, trimethyl amine, triethyl amine, methyl amine, ethyl amine, pripyl amine, butyl amine, diethyl amine, dimethyl amine and aniline. The most prefers amine for use in the present invention is triethyl amine.

Deprotection of the hydroxyl protecting groups of the cytidine compound of formula (XIII) is achived with aqueous ammonia in the presence of a Ci_-4 alcohol at reflux temperature to give the p-2'-deoxy-2',2'-difluorocutidine of the formula (II). Preferably, the suitable alcohol for use herein is methanol.

(XIII) (II)

Gemcitabine base

To prepare a compound of formula (I), gemcitabine base solution was acidified utilizing concentrated halo acid, such as hydrochloric acid, to give gemcitabine hydrochloride.

Gemcitabine hydrochloride

Aspects of the present invention will be described with reference to the following examples which should not be considered to limit the scope of the invention. To the technical persons in the field, it should be understood that the following examples are only used to demonstrate the practicality of the invention. Other embodiments will be apparent to one skilled in the art from consideration of the specification and examples.

EXAMPLE 1

Preparation of 3',5'-Di-0-benzoyl-p-2'-deoxy-2',2'-difluorocytidine (compound XIII) with 15 molar equivalents of cytosine A 250 ml three-necked round bottom flask was equipped with a condenser and then charged with 18.25 g (0.164 mol) of cytosine, 1 .085 g (0.008 mol) ammonium sulfate, 35.1 1 ml (0.167, 1 .02 eq) HMDS and then with the addition of 9 ml of toluene the mixture was heated to 125°C with slow stirring for a period of about 18 hours. Thereafter, 5 g (0.01 1 mol) of 2-Deoxy-2,2-difluoro-D-erythro- pentofuranose-3,5-dibenzoate-1 -a-methanesulfonate (compound X) in 30 ml of toluene solution was added dropwise thereto within 4.5 hours, after dropping completed, the mixture was stirred for 12 hours at the same condition(P:a=5: 1 ). Thereafter, the temperature of the reaction was cooled to room temperature and followed by the addition of 14 ml of toluene and then slow addition of 73 ml of water and finally 17.7 ml (0.21 mol) of concentrated hydrochloric acid was added and then heated to 70°C and stirred for 2 hours and then stirred at room temperature for further 1 hour. The precipitate was filtered and successively washed with 15 ml of 5% HCI and then with 15 ml of water to obtain a compound of formula (XII). The filtered precipitate was dissolved in 50 ml of methanol at reflux temperature with stirring and then with the addition of triethyl amine the pH was adjusted to about 7 and simultaneously the mixture was cooled to room temperature and stirred for 3 hours. The precipitate was filtered and washed with 10 ml of methanol and dried to yield 2.58 g of pure 3',5'-Di-O-benzoyl-p-2'-deoxy- 2',2'-difluorocytidine (50% yield) with the purity of greater than 99%. The product was characterized by comparison of its melting point, UV, IR spectra and HPLC graph/s with those previously reported for 3',5'-Di-O-benzoyl-p-2'-deoxy-2',2'- difluorocytidine.

EXAMPLE 2

Preparation of gemcitabine hydrochloride (compound I)

3',5'-Di-O-benzoyl-p-2'-deoxy-2',2'-difluorocytidine (2.58 g, 0.0055 mol) was suspended in 20 ml methanol and stirred at reflux temperature. Then 3.23 ml ammonium hydroxide 18.7% (0.0164 mol, 3 eq) was added to the resulting suspension and stirred at reflux temperature for about 6 hours. Thereafter, the resulting homogenous light brown solution was cooled to room temperature and treated with 0.258 g of active charcoal and stirring maintained for further 1 hour. The charcoal mixture was filtered over a pad of Celite and the resulting light yellow filtrate was evaporated under reduced pressure until about 8 ml of the solvent was remained. The resulting solution was acidified to pH 3 with concentrated HCI and then the precipitate was stirred at room temperature for 12 hours and thereafter the white precipitate was filtered and washed with 2 ml of methanol and dried to yield 1 .4 g of pure gemcitabine hydrochloride (85% yield) with the purity of greater than 99.8%. The product was characterized by comparison of its melting point, UV, IR spectra and HPLC graph/s with those previously reported for gemcitabine hydrochloride.

Claims

What is claimed:

1 . A process for preparing gemcitabine, which includes the following reaction:

(V) (IV) (VI)

Wherein, the substitution of R¹ is selected from the group consisting and not limited to benzoyl, nitrobenzyl, chloroacetyl, phenylbenzoyl, t-butyldiphenyl silyl, phenoxy acetyl, isobutyryl, ethoxy carbonyl, benzyloxy carbonyl, mesyl, trimethylsilyl, isopropyl dimethylsilyl, methyldiisopropyl silyl, triisopropyl silyl, t- butyldimethyl silyl, cinnamoyi, trityl, phenylcarbamoyi, phenoxycarbonyl, or naphtoyl, preferably, benzoyl. X is selected from the group comprising alkylsulfonyl, arylsulfonyl, substituted alkylsulfonyl, substituted arylsulfonyl, preferably, methanesulfonyl. R² is independentaly silyl protecting group and selected from the group consisting of CrC₇ trialkylsilyl, preferably, trimethylsilyl and R³ is H or acyl group

2. The process defined in claim 1 , wherein, the said reaction is: the alpha anomer enriched formula V compound is coupled during a glycosylation reaction to obtain a compound of formula VI.

3. The process defined in claim 1 , wherein, R¹ is dibenzoyl.

4. The process defined in claim 1 , wherein, R² is trimethylsilyl.

5. The process defined in claim 1 , wherein, R³ is hydrogen.

6. The process defined of any one of claims 1 to 5, wherein the said reaction is:

a compound of formula IV with certain molar ratio to the a-anomer enriched formula V is suspended in an inert organic solvent, and heated to 1 10- 130°C. Thereafter, the a-anomer enriched formula V is dissolved in an inert organic solvent and then added dropwise thereto within 4.5 hours, and then stirred for further 10 hours.

7. The process defined in claim 6, wherein, suitable inert water-immiscible organic solvents that can be employed include halogenated e.g. chlorinated hydrocarbons, e.g. dichloromethane, chloroform, tetrachloroethylene and 1 ,2-dichloroethane; esters e.g. (Ci_-4) alkyl esters e.g. ethyl acetate; ethers e.g. diisopropylether; aromatic hydrocarbons eg. toluene, xylenes etc. aromatic hydrocarbons are preferred.

8. The process defined in claim 6, wherein, the said temperature is preferably 125°C.

9. The process defined in claim 6, wherein, the said inert water-immiscible solvent is toluene.

10. The process of any one of claims 1 to 6, which further includes the following selectively isolating of a compound of formula VIII:

1 1 . The process defined in claim 10, wherein, R¹ , R² and R³ are the same as described in claim 1 .

12. The process defined in claim 10, wherein, R¹ is dibenzoyl.

13. The process defined in claim 10, wherein, the said alkyl amine is selected from the group comprising C5-C12 hetrocyclic amines and amines having the general formula R⁴R⁵R⁶N wherein R^4, R⁵ and R⁶ can be the same or different and are selected from the group comprising hydrogen, a CrC₆ alkyl group and a C6-C9 aryl group, with the proviso that the each of R^4, R⁵ and R⁶ are not hydrogen. Thus, it will be appreciated that the use of ammonia (i. e. R4=R5=R6=H) is outside the scope of the present invention. Non-limiting examples of suitable hetrocyclic amines include pyridine and piperidine. Non-limiting examples of other amines suitable include t- butylamine, trimethyl amine, triethyl amine, methyl amine, ethyl amine, propyl amine, butyl amine, diethyl amine, dimethyl amine and aniline.

14. The process defined in claim 1 0, wherein, the solvent is used to isolate the titled compound, is selected from the group comprising alcohol, ketone, ester, amide and nitril. Preferably, the solvent is selected from the group comprising ethanol, isopropanol, methanol, ethyl acetate and acetone.

1 5. The process defined in claim 1 0, wherein, the said alkyl amine is triethyl amine.

1 6. The process defined in claim 1 0, wherein, the said solvent is methanol.

1 7. The process defined in any one of claims 1 to 1 0, which further includes that the compound of formula VIII is deprotected in aqueous ammonia/Ci_-4 alcohol at reflux temperature to give a compound of formula II according to the following reaction:

1 8. The process defined in claim 1 7, wherein, the said alcohol is methanol.

1 9. The process defined in claim 1 7, wherein, NH₄OH/Compound (VIII) molar ratio is ranging from 2-4 and preferably the said ratio is 3.

20. The process defined in any one of claims of 1 to 1 7, which further includes that gemcitabine is converted to its corresponding hydrochloride salt utilizing hydrochloride acid to produce gemcitabine hydrochloride of formula I: