FORM-2
The Patent Act, 1970
(39 of 1970)
PROVISINAL SPECIFICATION
(Section 10; Rule 13)
Title; - Process for the preparation of Gemcitabine and its intermediates using Novel protecting groups and ion exchange resins,
Benzochem Life Sciences Pvt. Ltd, Vishwam, 8/B Postal Colony, Chembur, Mumbai- 400 071.
The following specification describes the nature of this invention and the manner in which it is to be performed: -

Title:
Process for the Preparation of Gemcitabine and its intermediates using Novel Protecting
groups and Ion Exchange Resins.
Inventors:
Kannan Vishwanath, Vikas Sadvilkar and Kashi Vishwanathan.
Inventor company:
Benzochem Lifesciences Pvt. Ltd.
Abstract:
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A process is outlined for the synthesis or preparation of gemcitabine hydrochloride with the help of a novel protecting group followed by the use of novel ion exchange resins to isolate and also to convert the gemcitabine base into the hydrochloride salt.


GEMCITABINE PATENT - SITUATION
EP0306190 (expiry 2008) Patent claims a process for preparing an enantiomeric mixture of erythro and threo lactones using a dioxolan in presence of a strong acid followed by azeotropic distillation of water. EP0576229 (expiry 2013)This patent protects a process for preparing an alpha-anomer enriched 2-deoxy-2',2-difluoro-Dribofuranosylsulfonates. EP0576232 (expiry 2013) Patent claims a process for separating 2-deoxy-2',2'-difluoro-D-ribofuranosylalkyl sulfonate anomers. EP0577302 (expiry 2013)This process provides an alpha-anomer enriched ribofuranosyl sulfonate from a beta-anomer ribofuranosyl sulfonate. EP0577303 (expiry 2013) This process provides a process for preparing a beta-anomer enriched nucleoside from an alphaanomer enriched carbohydrate based on a SN2-Displacement reaction. 6. EP0640614 (expiry 2014) Patent claims a stereoselective process for preparing a beta-enriched carbohydrate. The schematic synthesis does not reflect whether alpha or beta anomers or an enantiomeric mixture are used or a SN1 or SN2 displacement is favoured. EP0655454 (expiry 2014) We cannot evaluate this patent which claims a process for preparing useful intermediates for carbohydrates synthesis. EP0677527 (expiry 2015)This patent claims a process for preparing intermediates in carbohydrate synthesis. EP0688782 (expiry 2008)This patent claims a process for selectively isolating (erythro)-2',2'-difluorolactones from an enantiomeric mixture of erythro and threo lactones. EP0688783 (expiry 2008) Patent comprises a process for producing a 2'-deoxy-2',2'-difluoronucleoside in about a 1:1 alpha to beta ratio. EP0712860 expiry 2015) Patent claims a process to purify and isolate a beta-anomer enriched nucleoside in presence of a nucleobase and acetonitrile. EP0735043 (expiry 2013) It is not clear whether the patent also claims the introduction of fluor in the carbohydrate.
BACKGROUND OF THE INVENTION
Gemeitabine HCI, marketed by Eli Lilly under the trademark Gemzar<(R)>, is a nucleoside analogue that exhibits antitumor activity and belongs to a general group of chemotherapy drugs known as antimetabolites. Gemeitabine prevents cells from producing DNA and RNA by interfering with the synthesis of nucleic acids, thus stopping the growth of cancer cells and causing them to die.
Gemeitabine is a synthetic glucoside analog of cytosine, which is chemically described as 4-amino-l-(2-deoxy-2,2-difluoro-[beta]-D-ribofuranosyl)-pyrimidin-2(IH)-one or T- deoxy-2',2'-difluorocytidine ([beta] isomer). Gemeitabine HCI has the following structure:
Gemzar is supplied in vials as the hydrochloride salt in sterile form, only for intravenous use, containing either^f^mgjDr J g of gemeitabine HCI (as free base) formulated with mahnitdl (Jjfttf.mg o? 1 g, respectively) and sodium acetate (12.5 mg or 62.5 mg, respectively) as a sterile lyophilized powder. Hydrochloric acid and/or sodium hydroxide may have been added for pH adjustment.
U.S. Patent No. 4,808,614 ("the '614 patent") describes a process for synthetically producing gemeitabine.
D-glyceraldehyde ketal s is reacted with bromodifluoroacetic acid ethyl ester
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(BrCFaCOOEt) in the presence of activated zinc, to obtain ethyl 2,2-difluoro-3-hydroxy-3-
(2,2-dimethyldioxolan-4-yl)-propionate 3 as a mixture of 3-R and 3-S isomers. The 3-R to 3-
S isomer ratio is about 3:1. The 3-R isomer has the stereochemistry required for producing the desired erythro (3-R) ribose structure, and can be separated from the 3-S isomer by chromatography.
The resulting product is cyclized by treatment with an acidic ion exchange resin, such as Dowex 50W-X12, to produce 2-deoxy-2,2-difluoro-D-erythro-pentanoic acid-[gamma]- lactone 4. The hydroxy groups of the lactone are protected with fert-butyldimethylsilyl
(TBDMS) protecting groups to obtain the protected lactone 3,5-bis-(tert- butyldimethylsilyloxy)-2-desoxy-2,2-difluoro-l-oxoribose 5, and the product is reduced to obtain 3,5-bis-(tert-butyldimethylsilyl)-2-desoxy-2,2-difluororibose6.
The 1 -position of the carbohydrate is activated by the introduction of a leaving group, e.g., methanesulfonyloxy (mesylate), formed by reacting compound 6 with methanesulfonyl chloride to obtain 3,5-bis-(ter?-butyldimethylsilyloxy)-l-methanesulfonyloxy-2-desoxy-2,2-difluororibose 7. The base ring is coupled to the carbohydrate by reacting compound 7 with N,0-bis-(trimethylsilyl)-cytosine 8 in the presence of a reaction initiator, such as trifluoromethanesulfonyloxy trimethylsilane (trimethylsilyl triflate). Removal of the protecting groups and chromatographic purification affords gemcitabine free base.
U.S. Patent No. 4,526,988 describes a similar process in which the cyclization is carried out by hydrolyzing an alkyl 3 -dioxolanyl-2,2-difluoro-3 -hydroxy-propionate with an acidic ion exchange resin. See also, Hertel et al. in J Org. Chem. 53, No. 11, 2406 (1988). [0009] U.S. Patent No. 4,965,374 ("the '374 patent) describes a process for producing gemcitabine from an intermediate 3,5-dibenzoyl ribo protected lactone of the formula:
where the desired erythro isomer can be isolated in a crystalline form from a mixture of erythro and threo isomers.
The 3-hydroxy group of compound 3 is esterified with a benzoyl protecting group by reaction with benzoyl chloride, benzoyl bromide, benzoyl cyanide, benzoyl azide, etc. (e.g., PhCOX, wherein X = CI, Br, CN3 or N3), in presence of a tertiary amine or a catalyst such as 4-(dimethylamino)pyridine or 4-pyrrolidinopyridine, to obtain ethyl 2,2-difluoro-3- benzoyloxy-3-(2,2-dimethyldioxolan-4-yl)-propionate 9.
The isoalkylidene protecting group of 9 is selectively removed, e.g., by using a strong acid such as concentrated sulfuric acid in ethanol, to produce ethyl-2,2-difluoro-3- benzoyloxy-4,5-dihydroxypentanoate 9A. The product is cyclized to lactone 10 and converted to the dibenzoate ester to produce the lactone 2-deoxy-2,2-difluoropentofuranos-l- ulose-3,5-dibenzoate 11 as a mixture of erythro andthreo isomers. The '374 patent describes isolating at least a portion of the erytliro isomer from the mixture by selective precipitation. See also, Chou et al, Synthesis, 565-570, (1992). [0012] Compound 11 is then reduced to obtain a mixture of [alpha] and [beta] anomers of 2-desoxy- 2,2-difluorpentofuranose-dibenzoate 12, which is activated with methanesulfonyl chloride to obtain an anomeric mixture of mesylates, 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-di-0- benzoyl-1-0-[beta]-methanesulfonate 13, and coupled with N,0-bis(trimethylsilyl)-cytosine 8 to obtain the dibenzoate ester of the silyl-protected nucleoside 14 as a mixture of the [alpha]- and [beta]- anomers (about a 1:1 [alpha]/[beta] anomer ratio). Removal of the esters and silyl protecting group provides a mixture of the [beta]-anomer (gemcitabine) and the [alpha]-anomer (about a 1:1 [alpha]/[beta] anomer ratio). The '374 patent
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describes selectively isolating the [beta]-anomer (gemcitabine) by forming a salt of the anomeric mixture, e.g., the hydrochloride or hydrobromide salt, and selectively precipitating to obtain 2'-deoxy-2',2'-difluorocytidine as the salt in 1 :4 [alpha]/[beta] ratio. The '374 patent also describes selectively precipitating the [beta]-anomer in free base form in a slightly basic aqueous solution. One such process involves dissolving the 1:1 [alpha]/[beta] anomeric mixture in hot acidic water (pH adjusted to 2,5-5.0) and, once the mixture is substantially dissolved, increasing the pH to 7.0-9.0 and allowing the solution to cool, to produce crystals, which are isolated by filtration.
Processes for separating anomeric mixtures of alkylsulfonate intermediates also have been described. U.S. Patent Nos. 5,256,797 and 4,526,988 describe processes for separating anomers of 2-deoxy-2,2-difluoro-D~ribofuranosyl-l-alkylsulfonates, and U.S. Patent No. 5,256,798 describes a process for obtaining [alpha]-anomer-enriched ribofuranosyl sulfonates.
Other intermediates that may be useful for preparing gemcitabine have been disclosed. For instance, U.S. Patent No. 5,480,992 describes anomeric mixtures of 2,2- diiluororibosyl azide and corresponding amine intermediates that can be prepared, e.g., by reacting a 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-di-0-benzoyl-l-0-[beta]-methanesulfonate with an azide nucleophile, such as lithium azide, to obtain the azide. Reduction of the azide produces the corresponding amine, which can be synthetically converted into a nucleoside. See also U.S. Patent Nos. 5,541,345 and 5,594,155.
Other known intermediates include, e.g., l-alkylsulfonyl-2,2-difluoro-3- carbamoyl ribose and related nucleoside intermediates (U.S. Patent No. 5,521,294), tritylated intermediates (U.S. Patent No. 5,559,222), 2-deoxy-2,2-diflubro-[beta]-D-ribo-pentopyranose (U.S. Patent No. 5,602,262), 2-substituted-3,3-difluorofuran intermediates (U.S. Patent No. 5,633,367), and [alpha],[alpha]-difluoro-[beta]-hydroxy thiol esters (U.S. Patent Nos. 5,756,775 and
5,912,366).
Other processes for preparing gemcitabine have been described e.g., in WO
2006/070985 and WO 2006/071090, for stereoselective preparation of 2'-deoxy-2', T-difluorocytidine The process comprises activating the 2'-deoxy-2',2'-difiuoro-D-ribofuranosyl-3,5-diester 12A with diphenyl chlorophosphate followed by isomer separation, then obtaining the corresponding 1-bromo-ribofuranose intermediate, which is coupled withN.O-bis-(trimethylsilyl)-cytosine 8, then deprotected to obtain gemcitabine. However, this process requires an additional step as compared to the process outlined in Scheme 2, which makes it less attractive for industrial implementation. [0017] There are inherent problems associated with the production of gemcitabine, particularly for processes that require the production and separation of isomers, which tend to afford poor yields on a commercial scale. Accordingly, there is a need for improved methods of preparing gemcitabine and intermediates thereof, which facilitate the production of gemcitabine, particularly on a commercial scale. The present invention provides such methods and intermediates, as will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a process for preparing gemcitabine or a salt thereof, which preferably includes: removing at least a substantial portion of the [alpha] anomer from an anomeric mixture of a N-l-protected-2'-deoxy-2',2'-difluorocytidine-3',5'-diester to produce a product, which is at least enriched in the [beta] anomer; removing the 3 '-ester, the 5 '-ester and the N-protecting group; and optionally converting the product into a salt.
The preparation of the phosphate salt as the protecting group and the use of ion exchange resins for the isolation and the preparation of the hydrochloride salt conveniently is the major
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focus of this invention. The protected 2-deoxy-2,2-difluoro-D-erythro-pentofuranos-1 -ulose-3 ,5 -diesters of the present invention are particularly useful for the commercial scale production of gemcitabine. These D-erythro derivatives are crystalline materials, and can be purified by separation from a mixture of the D-erythro and L-threo isomers, e.g., by precipitation. The intermediates and methods of the present invention facilitate the total synthesis of gemcitabine, and are easier to use and produce higher yields than processes reported in the literature.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for preparing gemcitabine or a salt thereof, which preferably includes: Protection of the starting material in Step 1 with the help of an indigenous protecting group from the family of phosphate compounds, viz., Chloro dicresyl phosphoryl chloride. This compound is used to protect the hydroxyl group to facilitate further reactions at the desired sites.
Further the use of a novel catalyst, i.e., an ion exchange resin to isolate the product from Step
2 in presence of side products or impurities.
Finally the use of an ion exchange resin to convert the Gemcitabine base to the hydrochloride salt with the ease of operation involved therein.
The following example further illustrates the invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLE
This example demonstrates the preparation of crude 2-deoxy-2,2-difluoro-D- riboufuranose-
3 ,5-dibenzoate-1-arylate.
To a 500 ml round flask was added crude 2-deoxy-2,2-difluoro-D-erythro- pentofuranos-l-ulose-3,5-dibenzoate (23.36 mmol), which was dissolved in dry THF (100 ml) and cooled to 0-5C. Triethylamine (12.1 mmol) was added over a period of 5 minutes, after which time the mixture was stirred for another 1 hour. Chlorotricresyl phosphoryl chloride (23.36 mol) was added with stirring. Most of the solvents were distilled off, and to the residual volume (about 30 ml) IN HCI was added (50 ml) thus forming a clear solution with pH value of about 2. Ethyl acetate (100 ml) was added and the organic phase was washed with 5% NaHC03 (50 ml), water (50 ml), and brine (50 ml). The ethyl acetate phase was dried over MgS04 and concentrated under reduced pressure to obtain crude 2-deoxy-2,2-difluoro-D-riboufuranose~ 3,5-dibenzoate-1-arylate as an oil. Yield: 8.52g, (19.8 mmol, 84.8%, [alpha]/[beta] isomer ratio about 1/1).
In a 100 ml round flask, crude Step 1 ( 5.8mmol) was dissolved in dichloromethane (20 ml), and bis(trimethylsilyl)acetyl cytosine (0.7g, 6.9 mmol) was added. Triflate (0.79g, 6.9 mmol) was added dropwise while cooling to 0-5C. The mixture was stirred for another 1 hour, and washed with IN HCI (15 ml), 5% NaHC03 (15 ml), and dried over MgS04. The reaction mixture was stirred with an "Indion" Ion Exchange Resin to yield a product Step 2 Yield: 2.82 g, (5.55 mmol), 95.7%, purity 99.2%.
Ina 100 ml flask, the Step 2 product was diisolved in dry methanol 250 ml and stirred with an "Indion" Ion Exchange Resin 30 gms at 40C for 18-20 hours to afford Gemcitabine hydrochloride. Yield: 5 g, 50.5% yield. Purity: 98%, ee 95
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
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The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Date: July 16th 2007
To,
The Patent Officer.
The Patents and Trademarks Office,
Todi Industrial Estate,
Lower Parel, Mumbai.
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