PROCESS FOR PURIFYING AND ISOLATING 2'-DESOXY-2 ', 2'- DIFLUORONUCLEOSIDES
TECHNICAL FIELD The present invention relates to the field of pharmaceutical chemistry, and provides a process for purifying and isolating 2'-deoxy-2 ', 2'-difluoronucleosides. BACKGROUND TECHNIQUE The continued interest in the synthesis of 2'-deoxynucleosides and their analogs is reflected in their successful use as therapeutic agents in viral and cancerous diseases. A critical step in the total synthesis of the 2'-deoxynucleosides is the purification and isolation of the desired form of the nucleoside beta-anomer. This is critical, because the processes for the synthesis of 2'-deoxynucleosides are typically non-stereoselective, and form a mixture of alpha and beta nucleosides. Vorbruggen, et al., J. Org. Chem., 41, 2084 (1976); M. Hofer, Chem.
Ber. , 93, 2777 (1960); Walker et al., Nucleic Acid Reswarch, 12, 6827 (1984); R. P. Hodge et al., J. Org. Chem., 56, 1553 (1991); Tann et al., J. Org. Chem., 50, 3644 (1985); Howell et al., J. Org. Chem., 53, 85 (1988) and the patent of E.U.A. No. 4,965,374, by Chou et al., Report several syntheses of a mixture of the alpha and beta anomers of deoxy nucleosides. Despite advances in the process of nucleoside synthesis, there continues to be a need for a process capable of efficiently purifying and isolating REF: 24420 2'-deoxy-2 ', 2'-difluoronucleosides enriched in the beta anomer in increased yields , wherein the difluoronucleosides have been synthesized in the absence of a catalyst. Accordingly, the object of the present invention is to provide a process for efficiently purifying and isolating 2'-deoxy-2 ', 2'-difluoronucleosides enriched in the beta anomer. Other objects and advantages of the present invention will become apparent from the following description of the embodiments. DESCRIPTION OF THE INVENTION This invention is a process for purifying and isolating a nucleoside enriched in the beta anomer, comprising: a) providing a mixture containing R "and a nucleoside enriched in the beta anomer, of the formula
wherein each X is independently selected from hydroxy protecting groups, and R 'is a nucleobase of the formula:
wherein W is an amino protecting group; and R "is a nucleobase of the formula:
wherein W is an amino or hydrogen protecting group; in a high-bng solvent; b) diluting the mixture with an organic solvent selected from the group consisting of ethers, esters and nitriles; c) adding the diluted reaction mixture to aqueous acid; and d) keeping the acid mixture prepared in this manner at a temperature of from 70 ° C to 100 ° C, until the product of formula LB has precipitated, where W is now WAY TO CARRY OUT THE INVENTION throughout of this document, all temperatures are in degrees Celsius, all proportions, percentages and the like are by weight, and the units in all mixtures are in units of volume, except where otherwise indicated. The anomeric mixtures are expressed as a weight / weight ratio, or as a percentage. The term "alkyl" alone or in combination refers to straight chain, cyclic and branched aliphatic hydrocarbon groups, which preferably contain up to 7 carbon atoms, such as the methyl, ethyl, n-propyl, isopropyl, n-groups. butyl, t-butyl, n-pentyl, n-hexyl, 3-methylpentyl and the like, or substituted straight, cyclic or branched aliphatic hydrocarbons, such as chloroethyl, 1,2-dichloroethyl, and the like. The term "substituted", alone or in combination, refers to a substitution by one or more of the selected groups of cyano, halo, carboalkoxy, toluoyl, nitro, alkoxy, alkyl, and dialkylamino. The phrase "enriched in the anomer", alone or in combination, refers to an anomeric mixture wherein the ratio of a specified anomer is greater than 1: 1, and includes a substantially pure anomer. The hydroxy (X) protecting groups refer to hydroxy protecting groups known in the art, such as those described in Chapter 3 of Protective Groups in Organic Chemistry, McOmie Ed., Plenum Press, New York (1973), and Chapter 2 of Protective Groups in Organic Synthesis, Green, T. John Wiley and Sons, New York (1981). Preferred hydroxy protecting groups are the ester forming groups, such as formyl, acetyl, substituted acetyl, propionyl, butynyl, pivaloyl, 2-chloroacetyl, benzoyl, substituted benzoyl, phenoxycarbonyl, methoxyacetyl; carbonate derivatives such as phenoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, vinyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl and benzyloxycarbonyl; alkyl ether-forming groups, such as benzyl, diphenylmethyl, triphenylmethyl, t-butyl, methoxymethyl, tetrahydropyranyl, allyl, tetrahydrothienyl, 2-methoxyethoxymethyl; carbamates, such as N-phenylcarbamate and N-imidazolylcarbamate; most preferred are benzoyl, mono-substituted benzoyl and disubstituted benzoyl, acetyl, pivaloyl, triphenylmethyl ethers, and most preferred is benzoyl. The amino protecting groups (W) are selected from the group consisting of silylamine-forming groups, such as trialkylsilyl, which include trimethylsilyl; isopropyldialkylsilyl, alkyldiisopropylsilyl, triisopropylsilyl, 1,1,3,3-tetraisopropyldisiloxanyl, t-butyldialkylsilyl and t-butyldiarylsilyl; carbamates such as t-butoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, and 4-nitrobenzyloxycarbonyl; formyl, acetyl, benzoyl and pivalamide; groups that. they form ethers, such as methoxymethyl, t-butyl, benzyl, allyl and tetrahydropyranyl; A preferred amino protecting group is trirnethylsilyl. The first step in the process of the claimed invention is to provide a mixture containing R "and a nucleoside enriched in the beta anomer of the formula:
wherein each X is independently selected from hydroxy protecting groups, and R 'is a nucleobase of the formula
wherein W is an amino protecting group; and R "is a nucleobase of the formula
wherein W is an amino or hydrogen protecting group; in a high boiling solvent. Such a mixture can be created in many different ways. Methods for synthesizing such a mixture are described and claimed in European Patent Application No. 93304817.5. Following certain processes (that do not use a catalyzer, using an excess of nucleobase R ", page 9 of EP
93304817. 5; and using a high boiling point solvent, page 10, lines 13-17) described in European Patent Application No. 93304817.5, it is possible to prepare the nucleosides enriched in the beta anomer, in an anomeric ratio of alpha to beta from above. from 1: 1 to less than or equal to 1: 9. A large excess of R "must be used to obtain these ratios The excess of R" is separated from the desired product by the process of the present invention. The high-boiling solvent is a solvent that has a boiling point above 70 ° C. The solvent with high boiling point is moderately polar, stable to acid and non-nucleophilic. Typical high-boiling solvents are aromatic haloalkyl, alkoxy and halo substituted aromatics solvents, and mixtures thereof. The preferred high-boiling solvents are 1,2-dichloroethane, 1,1,2-trichloroethane, glyma, diglyme, toluene, xylenes, anisole, dichlorobromomethane, chlorobenzene, dibromochloromethane, tribromomethane, dibromomethane, acetomitrile, propionitrile, dioxane and mixtures thereof, while the most preferred one is anisole. Once a mixture containing R "and the nucleoside enriched in the described beta anomer is provided, the process of the present invention proceeds as follows: First, the reaction mixture is diluted with an organic solvent with a boiling point greater than 60 ° C. Suitable solvents are from the classes of ethers, esters, and nitriles, the preferred examples being acetonitrile, ethyl acetate and tetrahydrofuran.The dilution is carried out at an elevated temperature, which may be the The organic solvent must be heated to an elevated temperature, too, and the temperature of both, the reaction mixture and the solvent should be in the range of 70 ° C to 110 ° C. The most preferred solvent is acetonitrile. The amount of organic solvent added is in the range from
1 mi to 5 ml per 1 gram of R "(protected or unprotected cytosine) used No particular maintenance period is required after dilution of the reaction mixture, the diluted mixture can be taken immediately to the next stage. The diluted reaction mixture is added to a large amount of aqueous acid at an elevated temperature The purpose of the aqueous acid is to dissolve the excess R "(protected or unprotected cytosine), which was used in the glycosylation reaction described in page 9 of the application of EP No. 93304817.5. Therefore, the amount and degree of acidity of the aqueous acid depends on the excess amount of R "(protected or unprotected cytosine) used in the reaction itself.In addition, the amount of aqueous acid also depends on the selection of the substance. acid used to prepare the aqueous acid The most preferred acid is hydrochloric acid, used in a concentration from 1 N to 6 N, more preferably 4 N. When this acid is used, and the amount of excess R "(cytosine protected or unprotected) is in the range from 5X to 20X, the amount of aqueous hydrochloric acid is from 3 ml to 5 ml per 1 gram of R "(protected or unprotected cytosine) used, however, other acids are also usable aqueous minerals and conditions, and may be preferred in various circumstances., mineral acid substances such as sulfuric acid, sulfurous acid, phosphoric acid, nitric acid, and phosphonic acid can be used if desired by the operator. The concentration of the acid can be varied rather widely, approximately in inverse proportion to the acceptable volume of the total isolation stage. In general, concentrations from 1 N to 10% in the aqueous acid can be used. The volume of aqueous acid must be optimized experimentally for the individual acid and the amount in the reaction mixture. The necessary experiments are very simple, requiring only that the operator make successive adjustments of the concentration and volume of the acid with the specific reaction mixture in use, observing the solubility of R "(protected or unprotected cytosine) in each case. Aqueous acid does not need to be hot when combined with the reaction mixture.The aqueous acid can be at room temperature, provided that the entire mixture is heated to a temperature in the range of 70 ° C to 100 ° C. it may be sufficient to bring the reaction mixture to that temperature, or it may be necessary in some cases to heat the mixture externally.In some cases, the heat of reaction is greater than 100 ° C, and thus, the reaction mixture must be cooled , so that it remains below 100 ° C. This is so because it is important that the temperature of the aqueous acid is not high that any of the protective groups is hydrolyzed prematurely at this stage of the process. When acetonitrile is used as the organic solvent, the most highly preferred temperature is from 70 ° C to 80 ° C. The acid mixture resulting from the addition of the diluted reaction mixture to the aqueous acid is maintained, preferably with moderate agitation, by a period of time The physical changes that occur during this maintenance period are the dissolution of the excess of R "(protected or unprotected cytosine) in the aqueous acid layer, and the precipitation of the desired beta-nucleoside, the precipitation is selective, and the alpha-nucleoside Undesirable remains largely dissolved in the organic layer.Thus, the acid mixture must be kept at a constant temperature, until these two physical changes have occurred.In general, a period from 10 minutes to 1 hour is adequate. With sufficient maintenance period, the precipitated beta-nucleoside is separated from the two liquid phases by filtration or centrifugation, and washed with additional aqueous acid.The filtration or centrifugation should be carried out at an approximately constant temperature, to prevent the R "Dissolved (protected or unprotected cytosine) is separated by precipitation from the solution.
The beta nucleoside isolated and purified in this manner may still have an amino protecting group (W) present, or the amino protecting group (W) may have been hydrolyzed, and replaced with a hydrogen atom. The beta nucleoside isolated and purified in the above manner is of superior purity, with respect to the alpha nucleoside, R "(protected or unprotected cytosine) and other impurities, and it is also found that the desired product is prepared in superior performance When the acid mixture has been filtered or centrifuged to extract the solid product, the organic and aqueous layers are separated from the filtrate.The excess of R "(protected or unprotected cytosine) is in the aqueous layer, and can be extracted from that layer and recycled back to the process. The R "(protected or unprotected cytosine) can be recovered by simply cooling the aqueous layer and filtering the precipitated R (protected or unprotected cytosine), or by making the aqueous layer basic, cooling the basic solution, and filtering to collect the R "precipitate (protected or unprotected cytosine) The cytosine recovered from the above process is routinely recycled to the process of making the mixture, as described in EP No. 93304817.5 Thus, the claimed process provides an economical recycling of R" ( protected or unprotected cytosine), which is a beneficial property of the process. The final phase of the process is the elimination of the protective groups X and any remaining W of the solid, purified blocked nucleoside of formula IB. The same anomeric unprotected nucleoside ratio is obtained by the removal of the protecting groups. Most silyl-amino protecting groups are easily hydrolyzed by the use of a protic solvent, such as water or an alcohol. Most silyl-amino protecting groups are vulnerable to hydrolysis during their contact with a mineral acid. Acyl protecting groups, such as benzoyl and acyl-amino protecting groups, are removed by hydrolysis with a strong base, at a temperature from 0 ° C to 100 ° C. Strong or moderately strong bases suitable for use in This reaction is the bases that have a pKa (at 25 ° C) from 8.5 to 20.0. Such bases include alkali metal hydroxides, such as sodium or potassium hydroxide; alkali metal alkoxides such as sodium methoxide or potassium t-butoxide; alkali metal amides; amines such as diethyl amine, hydroxylamine, ammonia and the like; and other common bases such as hydrazine and the like. At least one base equivalent is needed for each protective group. The acyl protecting groups can also be removed with acid catalysts, such as methanesulfonic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, or with acidic ion exchange resins. It is preferred to carry out such hydrolysis at a relatively high temperature, such as the reflux temperature of the mixture, but temperatures as low as the environment can be used when particularly strong acids are used. Care must be taken to preserve these acyl protecting groups, so that they are not hydrolyzed prematurely during the early stages of the process of the invention. The removal of the ether protecting groups is carried out by known methods, for example, with ethanethiol and aluminum chloride. The protective group of t-butyldimethylsilyl requires acidic conditions, such as contact with a gaseous hydrogen halide, for its removal. The removal of the protecting groups can conveniently be carried out in alcoholic solvents, especially aqueous alkanols such as methanol. However, the deblocking reaction can also be carried out in any convenient solvent, such as polyoles including ethylene glycol, ethers such as tetrahydrofuran, ketones such as acetone and methyl ethyl ketone, or dimethylsulfide. In a preferred embodiment, the deblocking reaction employs ammonia to remove a benzoyl protecting group of hydroxy, at a temperature of 10 ° C. It is preferable, however, to use an excess of base in this reaction, although the amount of excess base used is not crucial. The nucleoside enriched in the resulting beta anomer
or an organic or inorganic acid addition salt thereof, can be extracted and / or isolated from the reaction mixture by the process described in the patent of E.U.A. No. 4,965,374. The following example strates specific aspects of the present invention, and is not intended to limit the scope thereof in any respect, and should not be construed in this manner. Example 1 Preparation, purification and isolation of l- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-ammopyrimidin-2-one enriched in the beta anomer with 22.5 equivalents of bis-trimethylsilylcytosine To a 250 ml three-necked flask were added 30 g of cytosine, 25 mg of ammonium sulfate and 150 ml of hexamethyldisilazane, and the mixture was heated to 125 ° C, and maintained for 30 minutes after the dissolution of all solids. Then, the temperature was raised to 145 ° C, held until the boiling stopped, and then kept at 120 ° C under vacuum until solids began to form above the level of the liquid in the flask. Then the mixture was cooled to 105 ° C, and 25 ml of anisole were added. In another 125 ml flask, 10 ml of anisole and 5.75 g of 2-deoxy-2,2-difluoro-3,5-dibenzoyl-D-ribofuranosyl-methanesulfonate were combined, and the mixture was heated until an homogeneous liquid. That liquid was added at constant temperature to the cytosine mixture, and the combined mixture was maintained at 100 ° C for 24 hours. A portion of 133 ml of 4 N hydrochloric acid was placed in a 500 ml flask. A 31.3 ml portion of acetomtril was added to the reaction mixture, and the diluted reaction mixture was then poured over the acid with constant stirring, while a cooling bath was applied to the 500 ml flask. The combined mixture was then stirred at 70 ° C for 10 minutes, and then filtered at a constant temperature. The wet cake was then suspended for 10 minutes at 70 ° C with 25 ml of 4N hydrochloric acid, and filtered again. The filter cake was suspended for 10 minutes at 70 ° C with 25 ml of deionized water, filtered, and the wet cake was suspended again at 70 ° C with 50 ml of deionized water. The pH of the aqueous suspension was brought to 7 with sodium bicarbonate, and the mixture was stirred for 10 minutes at 50 ° C or a higher temperature, and filtered again. The filter cake was suspended once more with 50 ml of deionized water at 70 ° C for 10 minutes, filtered, and the filter cake was dried and analyzed. It weighed 3.98 g, representing an isolated yield of 61 percent, and contained less than 1 percent of the unwanted alpha-anomer. The present invention has been described in detail, including preferred embodiments thereof. However, it wbe appreciated that those skd in the art, with consideration of the present disclosure, may make modifications and / or improvements on this invention that fall within the scope and spirit of the invention, as set forth in the following claims.
It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.
Having described the invention as above, property is claimed as contained in the following: