RU2131880C1 - Method of preparing beta-anomer enriched nucleosides - Google Patents

Abstract

FIELD: chemical industry. SUBSTANCE: method of preparing beta-anomer enriched nucleosides of formula (I):

wherein T is fluorine and R is nucleoside defined in claim 1. SN₂ substitution is carried out optionally in suitable solvent of sulfonyloxy groups (IV) from monomer- enriched carbohydrate of formula (II):

Description

 This invention relates to a process of stereoselective glycosylation to obtain 2'-deoxyfluoronucleosides and to intermediate products of this process.

 The ongoing interest in the synthesis of 2'-deoxyfluoronucleosides and their analogues is based on their successful use as therapeutic agents for the treatment of viral and cancer diseases. The compound of particular interest is gemcitabine; see European Patent Description No. 211354 and US Pat. No. 4,526,988. Since these compounds are beta nucleosides, there is a need to provide such compounds in high yield.

 An essential stage in the synthesis of 2'-deoxyfluoronucleosides is the condensation or glycosylation of the nucleobase and carbohydrate (carbohydrate) to form an N-glycosidic bond.

 However, the synthesis of 2'-deoxynucleosides, as a rule, are non-stereoselective and lead to the formation of mixtures of alpha and beta nucleosides. For example, the process of US Pat. No. 4,526,988 did not result in stereoselective production of 2-deoxy-2,2-difluoro-beta-nucleosides, but instead gave a 4: 1 anomeric ratio of α to β 2-deoxy-2,2-difluoro-nucleoside. Even the optimization of protective groups could not increase the ratio of alpha and beta beyond 1: 1; see US Pat. No. 4,965,374, which used carbohydrate with benzoyl protecting or blocking groups.

где T выбран из водорода или фтора, и R представляет нуклеозид, выбранный из группы, состоящей из

где R₁ выбран из группы, состоящей из водорода, алкила, замещенного алкила и галоида;
R₂ выбран из группы, состоящей из гидрокси, галоида, азидо, первичного амино и вторичного амино;
R₃ выбран из группы, состоящей из водорода, алкила и галоида;
R₄, R₅ и R₆ независимо выбраны из группы, состоящей из водорода, -OH, -NH₂, N (алкила), галоида, алкокси и тиоалкила;
R₇ выбран из группы, состоящей из водорода, галоида, циано, алкила, алкокси, алкоксикарбонила, тиоалкила, тиокарбоксамида и карбоксамида;
Q выбран из группы, состоящей из CH, CR₈ и N;
где R₈ выбран из группы, состоящей из галоида, карбоксамида, тиокарбоксамида, алкоксикарбонила и нитрила, включающий SA₂ нуклеофильное замещение сульфонилокси группы (Y) обогащенного альфа-аномером карбогидрата формулы:

где X независимо выбран из гидроксизащитных групп, и
T имеет значения, определенные выше; по крайней мере молярным эквивалентом нуклеооснования (R''), выбранного из группы, состоящей из

где R₁-R₇ и Q имеют значения, определенные выше;
Z представляет гидроксизащитную группу (т.е. группу, защищающую гидроксигруппу);
W представляет аминозащитную группу; и
M^⊕ представляет катион;
деблокирование с образованием соединения формулы (I).The present invention provides a stereoselective glucosylation process to produce a beta-anomer-enriched nucleoside of the formula:

where T is selected from hydrogen or fluorine, and R is a nucleoside selected from the group consisting of

where R ₁ selected from the group consisting of hydrogen, alkyl, substituted alkyl and halogen;
R ₂ selected from the group consisting of hydroxy, halogen, azido, primary amino and secondary amino;
R ₃ selected from the group consisting of hydrogen, alkyl and halogen;
R ₄ , R ₅ and R _{6 are} independently selected from the group consisting of hydrogen, —OH, —NH ₂ , N (alkyl), halogen, alkoxy, and thioalkyl;
R _{7 is} selected from the group consisting of hydrogen, halogen, cyano, alkyl, alkoxy, alkoxycarbonyl, thioalkyl, thiocarboxamide and carboxamide;
Q is selected from the group consisting of CH, CR ₈ and N;
where R _{8 is} selected from the group consisting of halogen, carboxamide, thiocarboxamide, alkoxycarbonyl and nitrile, including SA ₂ nucleophilic substitution of the sulfonyloxy group (Y) of the alpha-anomer-rich carbohydrate of the formula:

where X is independently selected from hydroxy protecting groups, and
T has the meanings given above; at least the molar equivalent of the nucleobase (R ″) selected from the group consisting of

where R ₁ -R ₇ and Q are as defined above;
Z represents a hydroxy protecting group (i.e., a hydroxy protecting group);
W represents an amino protecting group; and
M ^⊕ represents a cation;
release to form a compound of formula (I).

 Throughout this description, all temperatures are presented in degrees Celsius, all proportions, percentages, and the like are expressed in weight units, and all mixtures in volume units, unless otherwise indicated. Anomeric mixtures are expressed as a weight / weight ratio or as a percentage.

The term "lactol" alone or in combination with others refers to 2-deoxy-2,2-difluoro-D-ribofuranose or 2-deoxy-2-fluoro-D-ribofuranose. The term “xylenes”, alone or in combination, refers to all xylene isomers and mixtures thereof. The term "carbohydrate" alone or in combination refers to activated lactol, in which the hydroxy group at the C-1 position is substituted with the desired leaving or leaving group. The term "halogen" alone or in combination refers to chlorine, iodine, fluorine or bromine. The term “alkyl” in itself in combination refers to straight, cyclic and branched chain aliphatic hydrocarbon groups which contain from 1 to 7 carbon atoms, and preferably contain up to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, 3-methylcentyl, and similar groups, or to substituted straight, cyclic or branched chain aliphatic hydrocarbon groups such as chloroethyl, 1,2-dichloroethyl and similarly. The term "alkoxy" alone or in combination refers to compounds of the general formula AO; where A represents alkyl. The term "aryl" alone or in combination refers to carbocyclic or heterocyclic groups, such as phenyl, naphthyl, thienyl and their substituted derivatives. The term "thioalkyl" alone or in combination refers to the general formula BS: wherein B is alkyl or hydrogen. The term "ester" alone or in combination refers to the general formula EOOC, wherein E is alkyl or aryl. The term “aromatic” alone or in combination refers to benzene / similar structures containing (4n + 2) delocalized π electrons. The terms "sulfonate" or "sulfonyloxy" alone or in combination refers to compounds of the general formula BSO ₃ , wherein B is alkyl, substituted alkyl, aryl or substituted aryl. The term "substituted" alone or in combination refers to the substitution of one or more groups selected from cyano, halogen, carboalkoxy, toluoyl, nitro, alkoxy, alkyl and dialkylamino. The phrase "enriched in anomer" alone or in combination refers to an anomeric mixture in which the ratio of specific anomers is higher than 1: 1 and includes a substantially pure anomer. The term "concentrated" alone or in combination refers to a solution in which the weight content of the carbohydrate dissolved in the solvent is higher than 20% by weight per unit volume of solvent. For example, dissolving 100 grams of carbohydrate in 200 milliliters of solvent will give a 50 percent solution of carbohydrate. The term “conjugated anion” refers to an anion of the general formula BSO ₃ -, where B is as defined above. The term "anomerization" alone or in combination refers to the epimerization at the C-1 position of a ribofuranolyl derivative.

где Y, X, T, R'' и R имеют значения, определенные выше.According to the present glycosylation process, the 2'-deoxy-2 ', 2'-difluoronucleosides and 2'-deoxy-2'-fluoronucleosides of formula (I) enriched in the beta anomer are prepared by reacting the alpha anomer-enriched carbohydrate of formula (II) with at least the molar equivalent of the nucleobase (R ″) and the release of the resulting nucleoside, as shown below

where Y, X, T, R ″ and R are as defined above.

It is believed that the glycosylation reaction proceeds via SN ₂ substituted (displacement). Consequently, the beta-anomer-enriched nucleoside products of the present invention are obtained stereoselectively by the reaction of a nucleobase with an alpha-anomer-enriched carbohydrate.

 Lactol starting materials suitable for use in the preparation of the alpha-anomeric enriched carbohydrate of formula (II) used in the present glycosylation process are known and freely synthesized using standard procedures commonly used by those skilled in the art.

где X представляет гидроксизащитную группу.For example, US Pat. No. 4,562,988, incorporated herein by reference, discloses the synthesis of 2,2-difluoro-2-deoxy-D-ribofuranose intermediates of the formula

where X represents a hydroxy protecting group.

где X представляет гидроксизащитную группу.In addition, the authors of Reichman et al., Cornohydr. Res., 42, 233 (1975) disclose the synthesis of 2-deoxy-2-fluoro-D-gibofuranose of the formula

where X represents a hydroxy protecting group.

 In a preferred embodiment of the present process, an alpha-anomer-enriched 2,2-difluoro-2-deoxy-D-ribofuranoso-3,5-dibenzoate intermediate of formula (III) is used.

A key core of the present invention is the discovery that a new alpha-anomer-enriched carbohydrate intermediate of formula (III) or (IV) can be reacted under nucleophilic substitution conditions that favor inversions (since SN ₂ ), giving enriched beta- an anomer of nucleosides of the formula (I).

 In order to achieve an effective reaction between the nucleobase and the alpha-anomer enriched carbohydrate of formula (II), a stereoselectively corresponding leaving group (V) must be attached to the lactol to activate lactol and generate an alpha-anomer enriched carbohydrate of formula (II). However, the particular selected deleted group depends on the selected nucleobase and the selected glycosylation reaction conditions.

 The preparation of an alpha-anomer-enriched carbohydrate intermediate of formula (II) is preferably carried out according to the procedure described in the two co-pending applications US 07/902301 and 07/902305.

Application No. 07/902301 describes a stereoselective process for preparing an α-anomer-enriched intermediate of formula (II), where T is fluorine, by reacting a lactol of formula (III) with an amine base having a pKa of 8 to 20 in a low-freezing inert solvent ; bringing the temperature of the reaction mixture to about -40 ... -120 ^o C; and adding a sulfonating reagent.

 The amine base is preferably selected from the group consisting of triethylamine, tributylamine, dibutylamine, diethylmethylamine, dimethylethylamine, benzylmethylamine, N-methylmorpholine, tripropylamine, dipropylethylamine, N, N-dimethylbenzylamine, diisorpropylethylamine, 1,8-diethylamino-diethylamine 2-diethylamine -7-ena, and 1,5-diazabicyclo- (4.3.0) non-5-ena. The amount of base, preferably used, ranges from about 1 molar equivalent to 2 molar equivalents and more, preferably from about 1.2 molar equivalent to 1.5 molar equivalents.

The reaction is carried out in an inert solvent having a freezing point preferably below -78 ^° C. Preferably, the solvents are selected from the group consisting of dichloromethane, 1,2-dichloroethane, dichorfluoromethane, acetone, toluene, anisole, chlorobenzene, and mixtures thereof.

The temperature of the solvent mixture is preferably below about -78 ^° C. For example, a compound of formula III in which X is benzoyl is added to dichloromethane and triethylamine at room temperature for 30 minutes. Then the reaction temperature decreased. ¹⁹ F NMR data was recorded at various temperatures and showed that as the temperature decreased, there was an increase in the α: β anomeric ratio of ionized lactol:
Temperature - Alpha / Beta Ratio
19 ^o C - 2.0: 1
-3 ^o C - 2.3: 1
-23 ^o C - 2.5: 1
-43 ^o C - 3.0: 1
-63 ^o C - 3.6: 1
-83 ^o C - 3.4: 1
Ionized lactol is then captured in solution at a lower temperature and higher alpha anomer ratio by the addition of a sulfonating (sulfonating) agent, forming the α-anomer-enriched carbohydrate of formula (II).

 Thus, by appropriate temperature selection, the ratio of α to β of the carbohydrate intermediate starting material can be varied.

 The leaving group (Y) is attached to the lactol via sulfonation. Sulphonating reagents are preferably selected from the group consisting of substituted and unsubstituted sulphonating alkyl halides, substituted and unsubstituted aryl sulphonating halides and anhydrides of alkyl sulphonic acids and anhydrides of aryl sulphonic acids, such as methanesulphonyl halide, ethanesulphonylhalogen halide, 2-chloro-l-p-benzene-halides -dinitrobenzenesulfonyl halide, bromobenzenesulfonyl halide, dibromobenzenesulfonyl halide, benzenesulfonic anhydride, p-bromobenzenesulfo anhydride acids and anhydride of methanesulfonic acid, and substituted and unsubstituted fluoroalkyl and fluoroaryl sulfonating halides and anhydrides of fluoroalkyl and fluoroaryl sulfonic acids, such as trifluoromethanesulfonyl anhydride, trifluoromethanesulfonyl halide, 1,1,1-trifluoroethanesulfonylhydrogen halide acid halide , octafluoro-substituted acid anhydride, anhydride and non-fluoro-substituted acid anhydride, depending on the desired leaving group; more preferred is methanesulfonyl halide. The α-anomer-enriched carbohydrate intermediates derived from ionized lactols, especially carbohydrates containing trifluoromethanesulfonyloxy, are unstable at room temperature and are therefore preferably reacted in situ with a nucleobase.

 Due to the reactivity of the sulfonating agents, it may also be desirable to carry out the glycosylation reaction in a batch or continuous manner for large scale operations.

 The α-anomer-enriched intermediates of formula (II) in which T is hydrogen can be prepared in a similar manner, except that lactol of formula (IV) is used as starting material.

где Y представляет сульфонат и каждый X независимо выбран из гидроксизащитных групп, источником сопряженного аниона сульфокислоты, при повышенных температурах, в инертном растворителе.U.S. Application 07/902305 describes yet another stereoselective process for preparing α-anomer-enriched intermediates of formula (II), where T is fluoro by treating the beta anomer of a ribofuranosyl sulfonate of the formula

where Y represents a sulfonate and each X is independently selected from hydroxy-protecting groups, a source of a conjugated sulfonic acid anion, at elevated temperatures, in an inert solvent.

The conjugated anion of sulfonic acid can be obtained using a number of techniques known to specialists in this field. They include:
(a) neutralizing an alkyl or aryl sulfonic acid such as 1-methanesulfonic acid, p-methylbenzenesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, p-bromobenzenesulfonic acid and camphorsulfonic acid, an alkali metal base such as potassium hydroxide, sodium hydroxide, sodium hydroxide, sodium hydroxide, potassium, sodium methylate and the like;
(b) neutralizing the alkyl or aryl sulfonic acids with an amine base such as triethylamine, trimethylamine, N, N-dimethylbenzylamine or N-methylmorpholine, or an aromatic nitrogen base such as pyridine.

Examples of conjugated sulfonic acid anions produced by this method include triethylammonium methanesulfonate, trimethylammonium methanesulfonate, N, N-dimethylbenzylammonium methanesulfonate, pyridinium methanesulfonate, triethylammonium (p-bromobenzene) sulfonate, tetraethylammonium (tetraethylammonium) (tetraethylammonium) (tetraethylammonium) (tetraethyl ammonium) sulfonate, pyridinium (p-toluene) sulfonate and pyridium-3-nitrobenzenesulfonate; triethylammonium methanesulfonate is more preferred; and finally
(c) a conjugated sulfonic acid anion can be prepared in situ by reaction of 2-deoxy-2,2-difluoro-D-ribofuranoses with sulfonic anhydride, such as benzenesulfonic anhydride, p-bromobenzenesulfonic anhydride or methanesulfonic anhydride, on a base such as triethylamine. The reaction products are, for example, 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-di-O-benzyl-1-methanesulfonate and triethylammonium methanesulfonate.

The beta-anomeric ribofuranosyl sulfonate and the conjugated sulfonic acid anion are heated at a temperature of from about 50 to 130 ^° C. or more, preferably to a reflux temperature of the solvent mixture.

 Solvents suitable for use in the anomerization process must be inert with respect to the reaction conditions; acetonitrile, 1,2-dichloroethene, 1,1,2-trichloroethane, chlorobenzene, bromobenzene, dichlorobromomethane, anisole, glyme, diglyme, methyl tert-butyl ether, tetrahydrofuran, dioxane, ethyl acetate, toluene, xylenes, pyridine, N- are preferred. methylpyrrolidinono, N, N-dimethylformamide, 1,3-dimethyl-2-imidazolidinone, N, N-dimethylacetamide, and mixtures thereof; most preferred are anisole, toluene, glyme, acetonitrile, and mixtures thereof.

 To increase the solubility and nucleophilicity of the metal salts used as the source of the conjugated sulfonic acid anion, a catalyst selected from crown ethers or phase transfer catalysts may be added; preferred catalysts are selected from 18-Crown-6, 15-Crown-5, 12-Crown-4 and tris [2- (2-methoxyethoxy) ethyl] amine.

 This process is carried out in atmospheric conditions and preferably anhydrous and essentially ends in a period of from about 15 minutes to 24 hours. The resulting α-anomer-enriched carbohydrates of formula (II) are obtained with an anomeric ratio of about 2.3: 1 to 3.0: 1 alpha to beta.

 The α-anomer-enriched intermediates of formula (II), where T is hydrogen, can be prepared in a similar manner, except that lactol of formula (IV) is used as starting material.

 Glycosylation reactions usually require the protection of the hydroxy groups of lactol before using them in order to prevent the reaction of their hydroxy groups with the nucleobase, or in any way decomposed. Hydroxy protecting groups (X) suitable for use in the present glycosylation process are groups independently selected from known protecting groups used in synthetic organic chemistry. Each selected hydroxy protecting group is preferably capable of being efficiently introduced into lactol and easily removed from it after the glycosylation reaction is completed. Hydroxy protecting groups known in the art are described in Chapter 3 of Protective Groups in Organic Chemistry, Mc Omie ed. Plenum Press; New York (1973) and Chapter 2 of Protective Groups in Organic Synthesis, Green, John, J. Wiley and Sons, New York (1981); ester forming groups such as formyl, acetyl, substituted acetyl, propionyl, butynyl, pivalamido, 2-chloroacetyl, benzoyl, substituted benzoyl, phenoxycarbonyl, methoxyacetyl are preferred; carbonate derivatives such as phenoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, vinyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl and benzyloxycarbonyl; alkyl ether forming groups such as benzyl, diphenylmethyl, triphenylmethyl, tert-butyl, methoxymethyl, tetrahydropyranyl, allyl, tetrahydrothienyl, 2-methoxyethyloxymethyl; and silyl ether-forming groups such as trialkylsilyl, trimethylsilyl, isopropyl dialkylsilyl, alkyldiisopropyloyl, triisopropylsilyl, tert-butyldialkylsilyl and 1,1,3,3-tetraisopropyldisiloxanyl; carbamates such as N-phenyl carbamate and N-imidazoyl carbamate; however, benzoyl, monosubstituted benzoyl and disubstituted benzoyl, acetyl, pivaloyl, triphenylmethyl ethers, and silyl ether forming groups, especially tert-butylmethylsilyl, are more preferred; with benzoyl being most preferred.

 When hydroxy-protecting groups are attached to lactol, the usual reaction conditions apply and depend on the nature of the particular hydroxy-protecting group selected. Typical reaction conditions are described in US Pat. No. 4,526,988.

 In accordance with the present process, at least an equimolar amount of nucleobase (R ″) relative to the amount of carbohydrate used is used. However, it is more preferable to use an excess of nucleobase in the range of from about 1 molar equivalent to 30 molar equivalents; more preferably from about 10 molar equivalents to 20 molar equivalents and most preferably from about 15 to 20 molar equivalents.

 The nucleobases used here (R ″) are commonly known to organic chemists and there is no need to discuss their synthesis. However, in order to be useful in the present glycosylation process, the nucleobases or their tautomeric equivalents that carry amino or hydroxy groups should preferably contain protecting groups, such as primary amino protecting groups (W) and / or hydroxy protecting groups (Z), depending from the nature of the nucleobase. Protecting groups prevent hydroxy or amino groups from allowing competitive reaction sites for the α-anomer-enriched carbohydrate. Protecting groups are attached to the nucleobase (R ") before it is reacted with the alpha-anomer-enriched carbohydrate of Formula II and then removed. The procedure for attaching the protective groups to the nucleobases is described in US Pat. No. 4,526,988.

 Preferred amino protecting groups (W) for the pyrimidine nucleobases are selected from the group consisting of silyl ether-forming groups such as trialkylsilyl, tert-butyldialkylsilyl and tert-butyldiaryl silyl; carbamates such as tert-butoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl and 4-nitrobenzyloxycarbonyl; formyl, acetyl, benzoyl and pivalamido; ether forming groups such as methoxy methyl; tert-butyl, benzyl, allyl and tetrahydropyranyl; a more preferred group is trimethylsilyl. Preferred amino protecting groups (W) for the purine nucleobases are selected from the group consisting of alkyl carboxamides, haloalkyl carboxamides and aryl carboxamides, such as 2-trialkylsilylethoxymethyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, tert-butyl, tetrahydromethyltetramethyltetylamide, tetramethyrtetramethyl, methoxythiomethyl, trityl, pivalamido, tert-butyldimethylsilyl, tert-hexyldimethylsilyl, triisopropylsilyl, trichloroethoxycarbonyl, trifluoroacetyl, naphthoyl, formyl, acetyl; sulfonamides such as sulfonamido and arylsulfonamido, and pivalamido is more preferred. In addition to serving as an amino group protecting group, the beer protection group increases the solubility of obviously insoluble derivatives of purine nucleobases and directs the N-glycoside combination of purine bases to 9 regioisomer, as opposed to 7 regioisomer.

 Preferred hydroxy protecting groups (Z) for the pyrimidine nucleobases are selected from silyl ether forming groups such as trialkylsilyl; carbamates such as tert-butoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl and 4-nitrobenzyloxycarbonyl; carbocyclic esters such as formyl, acetyl and pivalamido; trimethylsilyl is preferred. Preferred hydroxy protecting groups (Z) for purine nucleobases are selected from the group consisting of ether forming groups such as benzyl, tert-butyl, trityl, tetrahydropyranyl, tetrahydrofuranyl, methoxymethyl, trityl; esters such as formyl, acetylpropionyl, pivalamido, benzoyl, substituted benzoyl; carbonates such as carbobenzoxy, tert-butoxycarbonyl, carbethoxy, vinyloxycarbonyl; carbamates such as N, N-dialkylcarboamoyl; trialkylsilyl ethers such as tert-butyltrimethylsilyl, tert-hexyldimethylsilyl, triisopropyl-silyl; pivalamido is more preferred.

 By providing protecting groups to the nucleobases of the present process, these protecting groups themselves can be protected. For example, N-acetylcytosine can be protected by trimethylsilyl to give bis-trimethylsilyl-N-acetylcytosine.

 In addition to the above, it is often advisable to convert any keto-oxygen atoms of the nucleobase into an enolic form. This makes the nucleobase more aromatic and enhances the reactivity of the nucleobase with respect to the alpha anomer-enriched carbohydrate of formula (II). It is most convenient to analyze keto-oxygen atoms and provide them with silyl protective groups.

 Although not essential, it is advisable that the reaction between the α- / anomer enriched carbohydrate of formula (II) and the nucleobase is carried out in a dry atmosphere, for example in dry air, nitrogen or argon. This is because some nucleobase derivatives, such as silylated nucleobase derivatives, are moisture sensitive.

 Any solvents used to prepare the nucleobase may be removed or mixed with the reaction solvent prior to the glycosylation reaction, provided that the mixture is inert with respect to the glycosylation reaction.

 In the case where the glycosylation reaction is carried out in a reaction solvent, the solvent must be inert with respect to the glycosylation reaction.

 However, as mentioned earlier, the particular solvent used for the reaction will depend on the conditions of the glycosylation reaction (e.g., reaction temperature, solvent), the group to be removed, and the nucleobase used.

The glycosylation reaction can be carried out at a temperature in the range of about 170 ^° C. to -120 ^° C. under atmospheric conditions and usually generally ends in about 5 minutes to 20 hours.

 This process can be monitored using procedures well known to those skilled in the art, such as high pressure liquid chromatography (HPLC) or thin layer chromatography, which can be used to detect the formation of a nucleoside product.

When the reaction is carried out in solution, it is preferred that a high boiling inert solvent and a solution having a carbohydrate concentration of at least 20 percent carbohydrate be used. A concentration of carbohydrate of about 20 to 70% is preferred; and most preferably from about 30 to 50%. A suitable reaction temperature range is from about 70 ^° C. to about 170 ^° C.

The high boiling solvent preferably has a boiling point above about 70 ^° C. and is selected from the group consisting of non-nucleophilic, aromatic, haloalkyl, alkoxy and halogen-substituted aromatic solvents, and mixtures thereof. Preferred solvents are 1,2-dichloroethane, 1,1,2-trichloroethane, glyme, diglyme, toluene, xylenes, anisole, dichlorobromomethane, chlorobenzene, dibromochloromethane, tribromomethane, dibromomethane, acetonitrile, propionitrile, dioxane and mixtures thereof; more preferred is anisole.

 The alpha-anomer-enriched carbohydrate of formula (II), used with a high boiling solvent, contains a sulfonyloxy group selected from alkylsulfonyloxy, substituted arylsulfonyloxy and substituted arylsulfonyloxy, such as methanesulfonyloxy, 2-chloro-1-phenyl-sulfonyloxy-sulfonyloxy-sulfonyloxy-sulfonyloxy-sulfonyloxy bromobenzenesulfonyloxy.

где R₁, R₃, Z и N имеют значения, определенные выше.The nucleobase (R ″), preferred for use with high boiling solvents, is a compound selected from the group consisting of

where R ₁ , R ₃ , Z and N are as defined above.

When the carbohydrate of formula (II) enriched with the α-anomer contains a fluorosulfonyloxy group, it is unstable at temperatures above room temperature. Therefore, glycosylation reactions using these sulfonyloxy groups should be carried out at a temperature equal to or lower than room temperature. When the glycosylation reaction proceeds under these conditions, the solvent should be low freezing. The preferred temperature range for the reaction is from 25 to -120 ^o C. In this case, the preferred solvents are selected from the group consisting of dichloromethane, 1,2-dichloroethane, dichlorofluoromethane, acetone, toluene, anisole, chlorobenzene, and mixtures thereof; dichloromethane is more preferred. However, the optimal temperature of the glycosylation reaction carried out at low temperatures depends on the group to be removed (V).

For example, when the group to be removed is trifluoromethanesulfonyloxy, a preferred reaction temperature range is from about -50 to 25 ^° C; more preferably, a temperature of from about -20 ^° C. to about 25 ^° C., however, when the group to be removed is 1,1,1-trifluoroethanesulfonyloxy, octafluorobutanosulfonyloxy or nanofluorobutanesulfonyloxy, a preferred reaction temperature ranges from about -20 to 25 ^° C. and a temperature of about from 0 to 25 ^o C.

где R₁, R₂, R₃, Z и W имеют значения, определенные выше.Nucleobases (R ″), preferred for use at low temperatures, are compounds selected from the group consisting of

where R ₁ , R ₂ , R ₃ , Z and W have the meanings defined above.

 The nucleobase (R ″) may optionally be converted to a metal cation salt to enhance its nucleophilic reactivity with the α- / anomer enriched carbohydrate of formula (II) (i.e., anionic glycosylation). These cationic salts of the nucleobases are prepared by adding a base to the nucleobase in the solvent.

The base may be selected from the group consisting of sodium tert-butylate, sodium hydride, sodium methylate, sodium ethylate, lithium hydride, potassium hydride, potassium hydroxide, potassium methylate, potassium ethylate and potassium t-butylate. Alternatively, the base may be selected from trialkylamine or tetraalkylammonium. Suitable inert solvents for the reaction may be selected from the group consisting of acetonitrile, dimethylformamide, dimethylacetamide, 1,3-dimethyl-2-imidazolidinone, tetrahydrofuran, sulfolane, N-methylpyrrolidinone, dimethyl sulfoxide and mixtures thereof. The solvent may be removed prior to the glycosylation reaction or mixed with a solvent for the glycosylation reaction, provided that the mixture is substantially inert with respect to the glycosylation reaction. Suitable reaction temperatures range from about 23 ^° C. to about 130 ^° C.

где R₁ - R₇, Q, Z, W и M⁺ имеют значения, определенные выше.The nucleobase (R ″) is preferably selected from the group consisting of the following

where R ₁ - R ₇ , Q, Z, W and M ⁺ are as defined above.

 The alpha-anomer-enriched carbohydrate of the formula (II) under these conditions contains a sulfonyloxy group selected from alkylsulfonyloxy, arylsulfonyloxy, substituted alkylsulfonyloxy, substituted arylsulfonyloxy, fluoroalkylsulfonyloxy and fluoro-arylsulfonyloxy, such as trifluoromethanes, trifluoromethanes, trifluoromethanes reflux fluorobutanesulfonyloxy, nanofluorobutanesulfonyloxy, methanesulfonyloxy, 2-chloro-1-ethanesulfonyloxy, toluenesulfonyloxy, n-nitrobenzenesulfonyloxy and p-bromobenzenesulfonyloxy.

As noted previously, fluoro-sulfonyloxy groups of formula (II) tend to be unstable at higher temperatures, and the above reaction with a nucleobase salt with a metal cation should be carried out using a low-freezing inert solvent with such groups. Preferred reaction temperatures range from about 25 to -120 ^o C.

The glycosylation reaction may also be carried out in the absence of a solvent (i.e., melt glycosylation). Obviously, the temperature used should be sufficient to convert the α-anomer-enriched carbohydrate intermediate of formula (II) and the nucleobase into a melt phase. Preferred reaction temperatures range from about 100 to 160 ^° C; however, a temperature of about 110 to 160 ^° C is more preferred; and most preferred is a temperature of from about 130 to 150 ^o C.

 Enriched α- / anomer carbohydrate of formula (II) in a condensation comprises a sulfonyloxy group selected from alkylsulfonyloxy, arylsulfonyloxy, substituted alkylsulfonyloxy and substituted arylsulfonyloxy such as methanesulfonyloxy, 2-chloro-1-ethanesulfonyloxy, toluenesulfonyloxy, p-nitrobenzenesulfonyloxy and p-bromobenzene - sulfonyloxy.

где R₁, R₃, Z и W имеют значения, определенные выше.A nucleobase (R ″) suitable for use under fusion conditions is preferably selected from the group consisting of

where R ₁ , R ₃ , Z and W have the meanings defined above.

 The present process may also be promoted by a catalyst. When a catalyst is used, it significantly reduces the amount of nucleobase required, increases stereoselectivity, reduces the cost of processing, increases productivity, simplifies the isolation of the product and reduces the required reaction temperature, making it possible to use less heat-resistant carbohydrates. Therefore, in the present process, it is desirable to use a catalyst, which is a salt containing a non-nucleophilic anion. Preferred are metal salts of groups IA, IIA or Quaternary ammonium salts. The catalyst must be soluble in the reaction solvent and highly ionized.

Preferred are salt catalysts selected from the group consisting of potassium, barium, cesium and trialkylammonium salts of trifluoromethanesulfonic acid, nanofluorobutane sulfonic acid, sulfuric acid, perchloric, nitric and trifluoroacetic acids; potassium or cesium salts of trifluoromethanesulfonic acid are more preferred. A suitable reaction temperature is in the range of about 50 to 100 ^° C.

 The solvent is preferably selected from polar non-nucleophilic solvents such as glyme, diglyme, anisole, acetonitrile, propionitrile, dioxane, and mixtures thereof; acetonitrile is more preferred.

 The α-anomer-enriched carbohydrate of formula (II) under catalytic conditions preferably contains a sulfonyloxy group selected from alkylsulfonyloxy and arylsulfonyloxy groups such as methanesulfonyloxy, 2-chloro-1-ethanesulfonyloxy, toluenesulfonyloxy, p-nitrobenzenesulfonyloxyloxy.

где R₁, R₂, R₃, Z и W имеют значения, определенные выше.The nucleobase (R ″) for use under catalytic conditions is preferably selected from

where R ₁ , R ₂ , R ₃ , Z and W have the meanings defined above.

 The final phase of the reaction sequence is the removal of the X, Z and / or W protecting groups (i.e., deprotection) from the blocked nucleoside of formula (I). After deprotection, the same anomeric nucleoside ratio is obtained.

Most silyl and silylamino protecting groups are easily cleaved off using a protic solvent such as water or alcohol. Acyl protecting groups such as benzoyl and acylamino protecting groups are removed by hydrolysis with a strong base at a temperature of from about 0 to 100 ^o C.

Strong or moderately strong bases for use in this reaction are bases which have a pKa (at 25 ^° C.) of about 8.5 to 20. Such bases include alkali metal hydroxides such as sodium or potassium hydroxide; alkali metal alcoholates such as sodium methylate or potassium tert-butylate; amides of alkali metals; amines such as diethylamine, glyoxylamine, ammonia and the like; and other common bases such as hydrazine and the like. For each protecting group, at least one base equivalent is required.

 Acyl protecting groups can also be removed using acid catalysts, such as methanesulfonic acid, hydrochloric, hydrobromic, sulfuric acids or using acidic ion-exchange resins. It is preferable to carry out such hydrolysis at relatively high temperatures, such as the reflux temperature of the mixture, but such low temperatures as ambient temperature may be used when particularly strong acids are used.

 The removal of ether protecting groups is carried out using known methods, for example, using ethanethiol and aluminum chloride.

 The tert-butyldimethylsilyl protecting group requires acidic conditions, such as contact with gaseous hydrogen halide, to remove it.

 Removal of the protecting groups can conveniently be carried out in alcoholic solvents, especially aqueous alcohols, such as methanol. However, the deprotection reaction can also be carried out in any convenient solvent, such as polyols including ethylene glycol, ethers such as tetrahydrofuran, ketones such as acetone and methyl ethyl ketone or dimethyl sulfoxide.

In a preferred embodiment, the deprotection reaction employs ammonia to remove the benzoyl protecting group of the hydroxy group at a temperature of about 10 ^° C. It is preferred, however, to use an excess of base in this reaction, although the amount of excess of base is not significant.

 In accordance with the present process, nucleosides enriched with a beta anomer are obtained at an anomeric ratio of α to β higher than from 1: 1 to 1: 9.

 The resulting β-anomer enriched nucleoside of formula (I) can be extracted and / or isolated from the reaction mixture as described in US Pat. No. 4,965,374, which is incorporated herein by reference.

 The following examples illustrate specific aspects of the present invention, but they are not intended to limit its scope in any way, and should not be construed as limiting.

 Example 1

 Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3'-, 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 10 equivalents of bis trimethylsilylcytosine.

Bio-trimethylsilylcytosine was obtained by combining 2.44 cytosine, 5.15 ml of hexamethyldisilazane and 580 mg of ammonium sulfate with 5 ml of xylene and heating the solution under reflux at 120 ^° C. for 1 hour. An additional 5 ml of hexamethyldisilazane was added to form a homogeneous solution, which was heated under reflux for 30 minutes. Xylenes and excess hexamethyldisilazane were removed and a gelatinous bio-trimethylsilylcytosine was formed. 5.6 g of bis-trimethylsilylcytosine was placed in 20 ml of xylenes. Xylenes were removed and bis-trimethylsilylcytosine was again placed in 20 ml of xylenes. Bis-trimethylsilylcytosine was evaporated to dryness and placed in 5 ml of xylenes. 1 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanosulfonate was reacted with a bis-trimethylsilylcytosine solution at 127 ^° C. for 3.5 hours.

 High performance liquid chromatography (HPLC) analysis confirmed the completion of the reaction.

To extract the nucleoside product, the reaction mixture was cooled to 60 ^o C, diluted in 100 ml of ethyl acetate and washed with 200 ml of 1 N. of hydrochloric acid. An emulsion formed, and the two layers that formed separated. The organic layer was washed successively with 100 ml of 5% sodium bicarbonate and 100 ml of a saturated solution of sodium chloride, then dried over magnesium sulfate. Quantitative HPLC analysis of the ethyl acetate layer showed that the yield of the blocked beta-anomeric nucleoside was 50%.

 The anomalous ratio of beta to alpha blocked nucleoside was 2.2: 1.

Example 2
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3'5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 5 equivalents of bis-trimethylsilylcytosine .

To 2.8 g of bis-trimethylsilylcytosine, 3 ml of xylenes was added and the solution was heated to 120 ^° C. until the bis-trimethylsilylcytosine was solubilized. 1 g of 2-deoxy-2,2-difluoro-D-ribofuranzil-3,5-dibenzoyl-1-α-methanesulfonate, dissolved in 2 ml of xylenes, was heated and reacted with a bis-trimethylsilylcytosine solution at 130 ^° C. within 16 hours. HPLC analysis confirmed the completion of the reaction. The anomalous ratio of beta to alpha blocked nucleoside was 1.1: 1.

 To extract the nucleoside product, the reaction mixture was diluted with 15 ml of ethyl acetate and washed with 150 ml of 1 N. of hydrochloric acid. An emulsion took place, and the two layers that formed were separated. The organic layer was washed successively with 100 ml of water and 100 ml of 5% sodium bicarbonate, then dried over magnesium sulfate. For a more accurate HPLC analysis, 1 ml of the organic layer was evaporated to dryness and placed in 1 ml of acetonitrile. Quantitative analysis of the HPLC of the organic layer in acetonitrile indicated that the yield of blocked beta-anomeric nucleoside was 36%.

Example 3
Preparation of 1- (2'-deoxy-2 ', 2-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched in beta anomer as the hydrochloride salt in 15 equivalents of bis-trimethylsilylcytosine.

Bis-trimethylsilylcytosine was prepared by combining 18.33 g of cytosine and 10 ml of anisole with 64.3 ml of N-methyl-N-cytosine- (trimethylsilyl) trifluoroacetamide and heating the solution at 80 ^° C for 30 minutes.

5.0 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate dissolved in 10 ml of anisole were reacted with a solution of bis-trimethylsilylcytosine at 105 ^° C for 5 hours. The anomalous ratio of beta to alpha blocked nucleoside was 5.4: 1.

To isolate the nucleoside product, the reaction mixture was cooled to 60 ^o C, diluted with 75 ml of ethyl acetate and washed with 200 ml of 1 N. of hydrochloric acid. A translucent solution containing solid particles was formed. The solution was heated to 60-70 ^° C for 15 minutes, filtered, and the separated solid was washed successively with 20 ml of ethyl acetate, then dried in a vacuum oven at 40 ^° C for 16 hours. The resulting nucleoside product weighed 4.0 g, so pl. 252 - 256 ^o C. Quantitative analysis of HPLC confirmed that the product was the hydrochloride salt of a blocked beta-anomeric nucleoside with a yield of 75 percent.

Example 4
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 10 equivalents of bis- trimethylsilylcytosine.

 Bis-trimethylsilylcytosine was prepared using the procedure described in example 1, except that 20 g of cytosine, 380 ml of hexamethyldisilazane, 1.18 g of ammonium sulfate and 48 ml of xylene were used. Bis-trimethylsilylcytosine was dissolved in 24 ml of xylene. 9.6 g of 2-deoxy-2,2-difluoro-D-ribufuranosin-3,5-dibenzoyl-1-toluenesulfonate in the ratio of alpha to beta 70: 30 was dissolved in 24 ml of xylenes and reacted with a solution of bis-trimethylsilylcytosine for 1 hour . HPLC analysis confirmed the completion of the reaction.

To extract the nucleoside product, the reaction mixture was cooled to 65 ^° C and 100 ml of ethyl acetate was added. The solution was kept at 65 ^o C and washed with 200 ml of 1 N. of hydrochloric acid. An emulsion was obtained, and the two layers that formed were separated. The organic layer was washed with 200 ml of 5% sodium bicarbonate, then dried over magnesium sulfate. The anomalous ratio of beta to alpha blocked nucleoside was 1.1: 1. Quantitative HPLC analysis indicated that the yield of the blocked nucleoside beta anomer was 27 percent.

 Example 5

 Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta-anomer with 20 equivalents of trimethylsilylcytosine.

Bis-trimethylsilylcytosine was prepared by mixing 10 g of cytosine with 175 ml of hexamethyldisilazane and 25 mg of ammonium sulfate in a nitrogen atmosphere and heating the solution at 120 ^o C for 2 hours. The mixture was cooled to 80 ^° C. and diluted with 100 ml ethyl acetate. Hexamethyldisilazane and ethyl acetate were subsequently atmospheric distilled off at a temperature of 145 ^o C. This procedure was repeated twice, then the resulting bis-trimethylsilylcytosan was added to 15 ml of anisole and cooled to 100-115 ^o C.

5.75 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α- methanesulfonate, dissolved in 10 ml of anisole, was stirred at 45 ^° C. until a homogeneous solution was formed, and was reacted with a solution of bis-trimethylsilylcytosine at 115 - 120 ^o C for 7 hours.

 HPLC analysis confirmed the completion of the reaction. The anomalous ratio of beta to alpha blocked nucleoside was 7.3: 1.

To isolate the nucleoside product, the reaction mixture was cooled to 88 ^o C, diluted with 34 ml of ethyl acetate and washed with 125 ml of 4 N. of hydrochloric acid. A suspension containing solid particles formed, mixed for 1.5 hours at 80 ^° C. and filtered. The filtrate was washed with 50 ml of 4 N. hydrochloric acid and dried in a vacuum oven at 45 ^o C. The resulting nucleoside product weighed 4.6 kg. Quantitative analysis of HPLC showed that the yield of the blocked beta-anomer of the nucleoside was 79.5 percent.

 Example 6

 Preparation of 1- / 2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl / -4-aminopyrimidin-2-one enriched beta-anomer hydrochloride salt with 20 equivalents bis-trimethylsilylcytosine.

Bis-trimethylsilylcytosine was prepared using the procedure described in example 5. The solution was cooled to 100 ^o C.

5.75 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate, dissolved in 10 ml of anisole, was stirred at 45 ^° C. until a homogeneous solution formed , and was subjected to interaction with a bis-trimethylsilylcytosine solution at 110 - 115 ^o C for 16 hours. HPLC analysis confirmed that only 3.9% of unreacted 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate remained. The anomalous ratio of beta to alpha blocked nucleoside was 7.2: 1.

To extract the nucleoside product, the reaction mixture was cooled and diluted with 69 ml of ethyl acetate at 65 ^° C. The reaction mixture was then combined with 185 ml of 4N. of hydrochloric acid. The mixture was refluxed for 1 hour at 78 ^° C. to form a suspension. The suspension was filtered, and the solid was washed with 60 ml of 4 N. hydrochloric acid and dried in a vacuum oven at 45 ^o C. The nucleoside product weighed 3.62 g. Quantitative HPLC analysis confirmed that the product was the hydrochloride salt of the blocked beta-anomer of the nucleoside in 64.2% yield.

 Example 7

 Preparation of 9- / 2'-deoxy-2 ', 2'-difluoro-3', 5-di-O-benzoyl-D-ribofuranosyl / -5-aminopurine enriched with beta anomer with 15 equivalents of bistrimethylsilyladenine.

Bis-trimethylsilyladenine was prepared by combining 7 g of adenine and 109 ml of hexamethyldisilazane with 250 mg of ammonium sulfate and heating the mixture at 100-115 ^o C for 8 hours. The solution was heated under reflux for an additional 30 minutes, and the excess hexamethyldisilazane was subsequently removed, and 14.5 g of bis-trimethylsilyladenine was dissolved in 3 ml of anisole. 1.58 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate was reacted with a solution of bis-trimethylsilyladenine at 105-110 ^° C for 24 hours.

 HPLC analysis confirmed the completion of the reaction.

To extract the nucleoside product, the reaction mixture was cooled to 30 ^o C, diluted with 50 ml of ethyl acetate and washed with 75 ml of 4 N. of hydrochloric acid. An emulsion formed, and the organic layer was separated and washed sequentially with 75 ml of 5% sodium bicarbonate and 75 ml of a saturated solution of sodium chloride, then dried over magnesium sulfate.

 The anomalous ratio of beta to alpha blocked nucleoside was 6: 1.

 In the table. 1 at the end of the description, it is shown how the concentration of the carbohydrate and the selected carbohydrate affect the anomeric ratio of the nucleoside product.

 / N / D / means not determined. Carbohydrates (carbo) are hydroprotected and include ... α- or β-OMS is alpha or beta-2,2-difluoro-2-deoxy-D-ribofuranosyl-3,5-dibenzoyl-1-methanesulfonate; β / or α-OTs is beta or alpha-2,2-difluoro-2-des-hydroxy-D-ribofuranosyl-3,5-dibenzoyl-1-toluenesulfonate; and α- or β- -OBs is alpha or beta-2,2-difluoro-2-deoxy-D-ribofuranosyl-3,5-dibenzoyl-1-bromobenzenesulfonate. 70: 30 α: β-OOTs carbohydrates were obtained by anomerizing β-OOTs with a salt of p-toluenesulfonic acid. The yields are given based on the amount of carbohydrate and were calculated by quantitative analysis of reverse phase HPLC, where the corresponding peak of the product solution was compared with the standard, 1- (2'-deoxy-2 ', 2'-difluoro-3' -, 5'- di-O-benzoyl-β- -D-ribofuranosyl) -4-aminopyrimidin-2-one, with the exception of case (a), which gives the yield of the isolated product. (G) The concentration of carbohydrate (Carbo.Conc.) Is expressed as a percentage of carbohydrate by weight (grams) per unit volume of solvent (milliliters). The nucleobase protecting group in each example is trimethylsilyl.

 Example 8

 Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 20 equivalent bis- trimethylsilylcytosine.

To 5.78 g of cytosine was added 112 ml of hexamethyldisilazane and 100 mg of ammonium sulfate. The solution was heated to 115-120 ^° C. for 1.5 hours with stirring, and the excess hexamethyldisilazane was subsequently removed. The mixture was cooled to 60 ^° C. and dissolved in 40 ml of 1,2-dichloromethane, forming a homogeneous solution of bis-trimethylsilylcytosine.

To 1 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-N-3,5-dibenzoate was added 10 ml of dichloromethane and 0.54 ml of triethylamine. This solution was stirred at 23 ^° C for 30 minutes, cooled to -78 ^° C and reacted with 0.57 ml of trifluoromethanesulfonyl anhydride in 0.50 ml of dichloromethane to form the 2-deoxy-2,2-difluoro-D- enriched alpha-anomer ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate intermediate in solution. Measures were taken to maintain the temperature of the reaction mixture below -65 ^° C. Analysis of ¹⁹ F nuclear magnetic resonance (NMR) of the 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate intermediate enriched in the alpha anomer at 65 ^o C gave the following data:
¹⁹ F NMR (300 MHz, CDCl ₃ ), δ -77 (s, 3F, CF ₃ SO ₂ -) - III (d, J = 257 Hz, 1F, alpha anomer), -122 (d, J = 242 Hz, 1F, beta anomer), -124 (d, J = 257 Hz, 1F, alpha anomer), -126 ppm, (d, J = 242 Hz, 1F, beta anomer). It should be noted that all shifts of ¹⁹ F NMR peaks are given relative to hexafluorobenzene, to which a frequency of -162.9 ppm is prescribed. The ¹⁹ F NMR spectrum also indicated a fluorine-proton combination, but the nature of these combinations was not determined.

The solution of the alpha-anomer-enriched 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate was reacted with a solution of bis-trimethylsilylcytosine at -65 ^° C and the reaction temperature was allowed to rise to 23 ^° C, and the title blocking nucleoside formed, which was confirmed by HRLC analysis. The anomalous ratio of beta to alpha blocked nucleoside was 1.9: 1.

To extract the nucleoside product from the reaction mixture, 100 ml of dichloromethane and 200 ml of 1N were added. of hydrochloric acid. The organic layer was separated and washed with 200 ml of 5% sodium bicarbonate. The organic layer was separated again and washed with 200 ml of saturated sodium chloride. The desired nucleoside product precipitated from the organic layer. Quantitative HPLC analysis showed a 42 percent blocked nucleoside beta anomer yield. ¹ H NMR (DMSO); δ = 4.74 (4'H), 4.79 (5'H), 5.84 (5H), 5.88 (3'H), 6.44 (1'H), 7.56 (NH ₂ ), 7.68 (6H). ¹³ C NMR (DMSO): δ = 63.46 (5'C), 71.80 (3'C), 75.71 (4'C), 84.64 (1'C), 95.12 (5'C), 121.86 (2 ' C), 141.93 (6'C), 154.48 (2C), 165.87 (4C).

Example 9
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 20 equivalents of bis- trimethylsilylcytosine.

A bis-trimethylsilylcytosine solution was prepared by suspending 5.78 g of cytosine in 75 ml of dichloromethane and adding 20.57 ml of N-methyl-N-trimethylsilyl trifluoroacetamide and cooling the resulting solution to -30 ^° C.

To 1 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 10 ml of dichloromethane and 0.55 ml of triethylamine. This solution was stirred at 23 ^° C. for 30 minutes, cooled to -78 ^° C. and reacted with 0.57 ml of trifluoromethanesulfonyl anhydride in 1 ml of dichloromethane to form 2-deoxy-2,2-difluoro-D-ribofuranosyl enriched in alpha anomer - 3,5-dibenzoyl-1-trifluoromethanesulfonate in solution. Measures were taken to maintain the temperature of the reaction mixture below -65 ^o C. Enriched with alpha-anomer 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate in the form of a solution was reacted with a solution of bis-trimethylsilylcytosine at -30 ^o C, forming the target blocked nucleoside, as confirmed by HPLC analysis. The anomalous ratio of beta to alpha blocked nucleoside was 2.3: 1.

 To extract the nucleoside product from the reaction mixture, 200 ml of 1N was added. of hydrochloric acid. The organic layer was separated and washed with 5% sodium carbonate. Quantitative analysis of the HPLC of the organic layer showed a blocked beta-anomeric nucleoside yield of 45%.

Example 10
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 20 equivalents of bis-trimethylcytosine.

A solution of bis-trimethylsilylcytosine was prepared using the procedure described in example 8, and was cooled to -15 ^o C.

To 1 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 10 ml of dichloromethane and 0.54 ml of triethylamine. This solution was stirred at 23 ^° C for 30 minutes, cooled to -78 ^° C and reacted with 0.57 ml of trifluoromethanesulfonyl anhydride in 0.5 ml of dichloromethane to form the alpha-anomer 2-deoxy-2,2-difluoro-D-ribofuranosyl enriched - 3,5-dibenzoyl-1-trifluoromethanesulfonate in solution. Measures were taken to maintain the temperature of the reaction mixture below -65 ^o C. Enriched with alpha-anomer 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate solution was reacted with a solution of bis-trimethylsilylcytosine at - 15 ^o C, forming the target blocked nucleoside, as confirmed by HPLC analysis. The anomalous ratio of beta to alpha blocked nucleoside was 2.3: 1.

To extract the nucleoside product from the reaction mixture, dichloromethane was removed, and the resulting residue was dissolved in 21 ml of anisole, then 40 ml of water, and then heated to 90 ^° C. The solids that formed were removed from the solution. The organic and aqueous layers were separated, and the organic layer was subsequently washed with an additional amount of 10 ml of water. The beta-anomeric nucleoside product precipitated from the organic layer. Quantitative HPLC analysis revealed a 58% yield of the blocked beta-anomer of the nucleoside.

Example 11
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 20 equivalents of bis- trimethylsilylcytosine.

A solution of bis-trimethylsilylcytosine was prepared by suspending 5.78 g of cytosine in 20 ml of dichloromethane and adding 20.57 ml of N-methyl-N-trimethylsilyl trifluoroacetamide in 10 ml of dichloromethane and cooling the resulting solution to 0 ^° C.

To 1 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 10 ml of dichloromethane and 0.55 ml of triethylamine. This solution was stirred at 23 ^° C. for 30 minutes, cooled to -78 ^° C. and reacted with 0.57 ml of trifluoromethanesulfonyl anhydride in 1 ml of dichloromethane to form 2-deoxy-2,2-difluoro-D-ribofuranosyl enriched in alpha anomer - 3,5- / dibenzoyl-1-trifluoromethanesulfonamide in solution. Measures were taken to maintain the temperature of the reaction mixture below -65 ^o C. Enriched with the alpha-anomer 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate in solution was reacted with a solution of bdis-trimethylsilylcytosine when 0 ^o C, forming the target blocked nucleoside, as confirmed by HPLC analysis. The anomalous ratio of beta to alpha blocked nucleoside was 2.5: 1.

 To extract the nucleoside product from the reaction mixture, 250 ml of 1N are added. of hydrochloric acid. The organic layer was separated and washed with 200 ml of 5% sodium carbonate. Quantitative analysis of the HPLC of the organic layer revealed a 49 percent blocked beta-anomeric nucleoside yield.

Example 12
Preparation of 1 (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-acetamidopyrimidin-2-one enriched with beta anomer with 10 equivalents of bis-trimethylsilyl -N- acetylcytosine.

56 ml of hexamethyldisilazane and 698 mg of ammonium sulfate were added to 4 g of N-acetylcytosine. This solution was heated to 115-120 ^o C for 4 hours with stirring, and the excess hexamethyldisilazane was subsequently removed. The mixture was cooled to -50 ^° C and dissolved in 50 ml of 1,2-dichloroethane. 1,2-dichloroethane was removed and the resulting solid residue was redissolved in 50 ml of 1,2-dichloroethane. The 1,2-dichloroethane was again removed and an oily residue formed. An oily residue was taken in 2.5 ml of 1,2-dichloroethane, forming a homogeneous solution of bis-trimethylsilyl-N-acetylcytosine. To 1 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 2 ml of dry dichloromethane. This solution was cooled to -78 ^° C and reacted with 0.55 ml of triethylamine and 0.58 ml of trifluoromethanesulfonyl anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate enriched in alpha in solution. Measures were taken to maintain the temperature of the reaction mixture below -65 ^° C. The solution of the alpha-anomer-enriched 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate was reacted with a solution of bis-trimethylsilyl-N -acetylcytosine at 23 ^o C.

The reaction mixture was stirred at -60 ^o C for one and a half hours, forming the target blocked nucleoside, as confirmed by HPLC analysis. The anomalous ratio of beta to alpha blocked nucleoside was 2: 1.

 To extract the nucleoside product from the reaction mixture, 50 ml of dichloromethane was added. The organic layer was separated and washed successively with 50 ml of 5% sodium bicarbonate, then 50 ml of 1 N. hydrochloric acid and 50 ml of saturated sodium chloride. Quantitative analysis of the HPLC of the organic layer showed a 15 percent yield of blocked beta-anomeric nucleoside.

Example 13
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 20 equivalents of bis- trimethylsilylcytosine.

To 5.78 g of cytosine was added 112 ml of hexamethyldisilazane in 50 mg of ammonium sulfate. The mixture was heated to 115-120 ^o C for 3 hours with stirring, and the excess hexamethyldisilazane was subsequently removed. This solution was then cooled to 27 ^° C. and a solid residue was formed which was placed in 35 ml of dichloromethane, forming a homogeneous solution of bis-trimethylsilylcytosine.

To 1 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 10 ml of dichloromethane and 0.54 ml of triethylamine. The solution was cooled to -78 ^° C and reacted with 0.57 ml of trifluoromethanesulfonyl anhydride in 0.50 ml of dichloromethane to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate enriched in solution. Measures were taken to maintain the temperature of the reaction mixture below -65 ^o C.

The alpha-anomer-enriched solution of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate was reacted with a solution of bis-trimethylsilylcytosine at 27 ^° C to form the target blocked nucleoside, as confirmed by HPLC analysis and indicated that 11 percent of the unreacted alpha-anomer-enriched 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate remained.

 The anomalous ratio of beta to alpha blocked nucleoside was 2.2: 1. Quantitative analysis of HPLC showed a blocked beta-anomeric nucleoside yield of 54%.

Example 14
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 20 equivalents of bis- trimethylsilylcytosine.

To 5.78 g of cytosine was added 112 ml of hexamethyldisilazane and 50 mg of ammonium sulfate. This solution was heated to 115-120 ^o C for 2 hours with stirring, and the excess hexamethyldisilazane was subsequently removed. The resulting oil was cooled to 23 ^o C, forming a solid residue, which was placed in 35 ml of dichloromethane, forming a homogeneous solution of bis-trimethylsilylcytosine, and cooled to 0 ^o C.

To 1 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 9 ml of dichloromethane and 0.54 ml of triethylamine. The solution was cooled to -78 ^° C and reacted with 0.57 ml of trifluoromethanesulfonyl anhydride in 0.50 ml of dichloromethane to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate enriched in solution. Measures were taken to maintain the temperature of the reaction mixture below -65 ^o C.

The alpha-anomer-enriched solution of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate was reacted with a solution of bis-trimethylsilylcytosine at 23 ^° C. to form the target blocked nucleoside, as confirmed by HPLC analysis. The anomalous ratio of beta to alpha blocked nucleoside was 2.2: 1.

 To extract the nucleoside product from the reaction mixture, the mixture was washed twice with 150 ml of 1 N. of hydrochloric acid. The organic layer was separated, washed with 150 ml of 5% sodium bicarbonate and washed again with 150 ml of saturated sodium chloride. Quantitative analysis of the HPLC of the organic layer showed a 49 percent blocked beta-anomeric nucleoside yield.

Example 15
Preparation of 1- (2'-deoxy-2 ',' 2-difluoro-3 ', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta-anomer with 30 equivalents of bis- trimethylsilylcytosine.

To 5.9 g of cytosine was added 112 ml of hexamethyldisilazane and 25 mg of ammonium sulfate. The solution was heated to 120-125 ^° C. for 3 hours with stirring, and the excess hexamethyldisilazane was subsequently removed. The resulting solid residue was placed in 35 ml of dichloromethane and cooled to 10 ^° C, forming a homogeneous solution of bis-trimethylsilylcytosine.

To 655 mg of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 0.55 ml of dichloromethane and 0.36 ml of triethylamine. The solution was stirred at 23 ^° C. for 30 minutes, cooled to -78 ^° C. and reacted with 0.35 ml of trifluoromethanesulfonyl anhydride in 0.50 ml of dichloromethane to form the 2-deoxy-2,2-difluoro-D-ribofuranosyl- enriched alpha-anomer 3,5-dibenzoyl-1-trifluoromethanesulfonate in solution. Measures were taken to maintain the temperature of the reaction mixture below -65 ^o C.

The solution of the alpha-anomer-enriched 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate solution was reacted with a solution of bis-trimethylsilylcytosine at 10 ^° C. to form the target blocked nucleoside, as confirmed by HPLC analysis . The anomalous ratio of beta to alpha blocked nucleoside was 2.7: 1. Quantitative analysis of HPLC showed a blocked beta-anomeric nucleoside yield of 60 percent.

Example 16
Preparation of 1- (2'-deoxy-2 ',' 2-difluoro-3 ', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 20 equivalents of bis- trimethylsilylcytosine.

To 5.78 g of cytosine was added 112 ml of hexamethyldisilazane and 50 mg of ammonium sulfate. The solution was heated to -115-120 ^o C for one and a half hours with stirring, and the excess hexamethyldisilazane was subsequently removed. The resulting solid residue was placed in 40 ml of 1,2-dichloromethane at 23 ^° C, forming a homogeneous solution of bis-trimethylsilylcytosine.

To 1 mg of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 10 ml of dichloromethane with 1.2 ml of triethylamine. The solution was cooled to -78 ^° C and reacted with 0.57 ml of trifluoromethanesulfonyl anhydride in 0.50 ml of dichloromethane to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate enriched in solution.

Measures were taken to maintain the temperature of the reaction mixture below -65 ^° C. The solution of the 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate-enriched alpha-anomer was reacted with a solution of bis-trimethylsilylcytosine at 23 ^o C, forming the target blocked nucleoside, as confirmed by HPLC analysis.

 The anomalous ratio of beta to alpha blocked nucleoside was 2.8: 1. Quantitative HPLC analysis showed a 50% yield of blocked beta-anomeric nucleoside.

Example 17
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 20 equivalents of bis- trimethylsilylcytosine.

To 5.78 g of cytosine was added 112 ml of hexamethyldisilazane and 50 mg of ammonium sulfate. The mixture was heated to 115-120 ^o C for 1.5 hours with stirring, and the excess hexamethyldisilazane was subsequently removed. The resulting solid residue was placed in 40 ml of dichloromethane at 23 ^° C, forming a homogeneous solution of bis-trimethylsilylcytosine.

To 1 mg of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 10 ml of dichloromethane and 0.54 ml of triethylamine. The solution was stirred at 23 ^° C for 30 minutes, cooled to -78 ^° C and reacted with 0.57 ml of trifluoromethanesulfonyl anhydride in 0.50 ml of dichloromethane, forming the 2-deoxy-2,2-difluoro-D-ribofuranosyl- enriched alpha-anomer 3,5-dibenzoyl-1-trifluoromethanesulfonate in solution. Measures were taken to maintain the temperature of the reaction mixture below -65 ^o C.

The solution of the 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate-enriched alpha-anomer was reacted with a solution of bis-trimethylsilylcytosine in solution at 23 ^° C. to form the target blocked nucleoside, as confirmed by analysis HPLC.

 The anomalous ratio of beta to alpha blocked nucleoside was 2.5: 1. Quantitative HPLC analysis showed a blocked beta-anomeric nucleoside yield of 68 percent.

Example 18
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 20 equivalents of bis- trimethylsilylcytosine.

 To 5.78 g of cytosine was added 5 ml of dichloromethane, 20.6 ml of N-methyl-N-trimethylsilyl trifluoroacetamide and 5 ml of dichloromethane, forming a homogeneous solution of bis-trimethylsilylcytosine.

To 1 mg of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 3 ml of dichloromethane and 0.55 ml of triethylamine. This solution was stirred at 23 ^° C. for 30 minutes, cooled to -78 ^° C. and reacted with 0.57 ml of trifluoromethanesulfonyl anhydride in 1 ml of dichloromethane to form 2-deoxy-2,2-difluoro-D-ribofuranosyl enriched in alpha anomer -3,5-dibenzoyl-1-trifluoromethanesulfonate in solution. Measures were taken to maintain the temperature of the reaction mixture below -65 ^o C. The solution of the alpha-anomer-enriched 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate was reacted with a solution of bis-trimethylsilylcytosine at 25 ^o C, forming the target blocked nucleoside, as confirmed by HPLC analysis. The anomeric ratio of beta to alpha blocked nucleoside was 2.5: 1.

 To extract the nucleoside product from the reaction mixture, 250 ml of 1N was added. of hydrochloric acid. The organic layer was separated and washed with 250 ml of 5% sodium carbonate. Quantitative analysis of the HPLC organic layer showed a 50 percent blocked beta-anomeric nucleoside yield.

 In the table. 2 (see the end of the description) shows how the solvent, the number of molar equivalents of pyrimidine-nucleoside derivatives affect the anomeric ratio and yield of the nucleoside product.

The carbohydrate used to produce blocked nucleosides in the table was the alpha-anomer 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate enriched in alpha. (N / D) means not determined. The yield data are presented based on the amount of carbohydrate and were calculated based on the results of a quantitative analysis of reverse phase HPLC, in which the peak of the corresponding product solution was compared with the standard. The protective group for the above nucleoside base is trimethylsilyl
Example 19
Preparation of 1- (2 -'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with the beta anomer.

Cytosine (12.0 g), hexamethyldisilazine (60 ml) and ammonium sulfate (10 mg) were heated under reflux at 125 ^o C for 30 minutes, forming a homogeneous solution. Hexamethyldisilazane was removed by distillation to form bis-trimethylsilylcytosine. 2-deoxy-2 ', 2'-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate (1.15 g) was reacted with bis-trimethylsilylcytosine (6.89 g, 10 equiv.) In anisole (2 ml ) and acetonitrile (3 ml) at 80 ^o C in the presence of potassium salt of nanofluoro-1-butanesulfonic acid (0.5 g) for 16 hours.

 HPLC analysis confirmed the completion of the reaction and indicated an in situ yield of 33 percent. The anomeric ratio of beta to alpha of the target compound was 3: 1.

Example 20
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with potassium sulfate.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate (1.15 g) was reacted with bis-trimethylsilicytosine (6.89 g, 10 equiv.), Obtained as described in example 19, in acetonitrile (2.0 ml) at 80 ^o C in the presence of potassium sulfate (0.5 g) for 72 hours.

 HPLC analysis confirmed the completion of the reaction and showed an in situ yield of 65 percent. The anomeric ratio of beta to alpha of the target compound was 4.7: 1.

Example 21
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one with tetrabutylammonium salt of trifluoromethanesulfonic acid enriched with the beta anomer.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate (0.29 ml) was reacted with bio-trimethylsilylcytosine (6.89 g, 10 eq.), Obtained as described in Example 19, in acetonitrile (3.0 ml) at 80 ^° C. in the presence of tetrabutylammonium salt of trifluoromethanesulfonic acid (1.5 mmol) (obtained in situ by treating tetrabutylammonium hydroxide (1.5 ml of a 1 molar solution in methanol) with trifluoromethanesulfonic acid (0.13 ml)), then distillation to remove methanol within 4 hours.

 HPLC analysis confirmed the completion of the reaction and showed an in situ yield of 45 percent. The anomeric ratio of beta to alpha of the target compound was 7.1: 1.

Example 22
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched in beta anomer with barium sulfate.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate (1.15 g) was reacted with bis-trimethylsilylcytosine (6.89 g, 10 eq.), Obtained as described in Example 19, in acetonitrile (3.0 ml) at 75 ^o C in the presence of barium sulfate (1.0 g) for 20.5 hours. HPLC analysis showed an in situ yield of 36 percent. The anomeric ratio of beta to alpha of the target compound was 11.2: 1.

Example 23
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta-anomer with cesium sulfate.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate (1.15 g) was reacted with bis-trimethylsilylcytosine (6.89 g, 10 equiv.), Obtained as described in Example 19, in acetonitrile (3.0 ml) at 75 ^° C. in the presence of cesium sulfate (1.0 g) for 21 hours.

 HPLC analysis showed an in situ yield of 24 percent. The anomeric ratio of beta to alpha of the target compound was 17.9: 1.

Example 24
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta-anomer with the cesium salt of trifluoromethanesulfonic acid .

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate (1.15 g) was reacted with bis-trimethylsilylcytosine (6.89 g, 10 equiv.), Obtained as described in Example 19, in acetonitrile (3.0 ml) at 75 ^° C in the presence of the cesium salt of trifluoromethanesulfonic acid (obtained in situ by treating 0.13 ml of trifluoromethanesulfonic acid with excess cesium carbonate) for 20.5 hours.

 HPLC analysis confirmed the completion of the reaction and showed an in situ yield of 65 percent. The anomeric ratio of beta to alpha of the target compound was 7.2: 1.

Example 25
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with the barium salt of trifluoromethanesulfonic acid .

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate (1.15 g) was reacted with bis-trimethylsilylcytosine (6.89 g, 10 eq.), Obtained as described in Example 19, in acetonitrile (3.0 ml) at 75 ^o C in the presence of barium salt of trifluoromethanesulfonic acid (obtained in situ by treating 0.13 ml of trifluoromethanesulfonic acid with excess barium carbonate) for 20.5 hours.

 HPLC analysis showed an in situ yield of 25 percent. The anomeric ratio of beta to alpha of the target compound is 14.4: 1.

Example 26
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with the potassium salt of trifluoromethanesulfonic acid.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate (2.3 g, 12.6 equiv.) Was reacted with bistrimethylsilylcytosine (16.1 g), obtained as described in example 19, in acetonitrile (8.0 ml) at 75 ^° C. and in the presence of a potassium salt of trifluoromethanesulfonic acid (0.26 ml) with potassium carbonate (1.0 g) for 45 hours. HPLC analysis indicates a yield of 69.8% in situ. The ratio of beta to alpha isomers in the target product is 7.2: 1.

Nucleoside extract, the reaction mixture is cooled to 70-80 ^o and combined with 40 ml of 4N hydrochloric acid. Quantitative HPLC analysis indicates that the yield of the isolated product is about 62.4%
Example 27
Preparation of a beta anomer enriched in 1- (2-deoxy-2,2-difluoro-3,5-O-benzoyl-D-ribofuranosyl) -4-amino-pyrimidin-2-one with the potassium salt of trifluoromethanesulfonic acid.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate (2.3 g) was reacted with bis-trimethylsilylcytosine (16.1 g, 12.6 equiv.), obtained according to example 19, in propionitrile (8 ml) at 90 ^o C and in the presence of potassium salt of trifluoromethanesulfonic acid (obtained in situ by processing trifluoromethanesulfonic acid (0.26 ml) with potassium carbonate (1.0 g) for 21 hours.

 HPLC analysis confirmed the completion of the reaction. The anomeric ratio of beta to alpha of the target compound was 6.7: 1.

To extract the nucleoside product, the reaction mixture was cooled to a temperature between 70 and 80 ^o C and combined with 40 ml of 4 N. of hydrochloric acid. The product precipitated, filtered and dried. Quantitative analysis of HPLC showed a yield of 59.3% of the isolated product.

Reference Example 28
Preparation of Enriched 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one without a catalyst.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate (1.15 g) was reacted with bistrimethylsilylcytosine (6.09 g, 10 equiv.), Obtained as described in example 19, in anisole (4 ml) at 110 ^o C for 20 hours.

 HPLC analysis confirmed the completion of the reaction and showed an in situ yield of 77 percent. The anomeric ratio of beta to alpha of the target compound was 3.4: 1.

Reference Example 39
Preparation of Enriched 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one without a catalyst.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate (1.15 g) was reacted with bistrimethylsilylcytosine (6.08 g, 10 eq.), Obtained as described in example 19, in propionitrile (4 ml) at 85 ^o C in the presence of cesium salt of trifluoromethanesulfonic acid (obtained in situ by processing trifluoromethanesulfonic acid (0.13 ml) with an excess of cesium carbonate) for 20 hours.

 HPLC analysis confirmed the completion of the reaction and showed an in situ yield of 70 percent. The anomeric ratio of beta to alpha of the target product was 6.7: 1.

Example 30
Preparation of Beta Anomer-Enriched 9- [1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl)] -2,6-dipivalamidoaminopurine with 2 equivalents potassium salt of 2,6-dipivalamidoaminopurine.

To 100 mg of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 1 ml of dichloromethane and 0.036 ml of triethylamine. This solution was stirred at 23 ^° C. for 15 minutes, cooled to -40 ^° C. and reacted with 0.045 ml of trifluoromethanesulfonic anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5- enriched with alpha-anomer dibenzoyl-1-trifluoromethanesulfonate in solution.

185 mg of a suspension of 2,6-dipivalamidoaminopurine was prepared in 1.5 ml of acetonitrile and maintained anhydrous in an atmosphere of azone. 65 mg of potassium tert-butylate was added to the suspension, and the resulting mixture was stirred at 23 ^° C. for 10 minutes to form the potassium salt of 2,6-dipivalamidoaminopurine. The salt was cooled to 0 ^° C and reacted with an alpha-anomer-enriched solution of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate, stirred for 1 hour and heated to 22 ^° C , forming the target blocked nucleoside, as confirmed by HPLC analysis.

 The anomalous ratio of beta to alpha blocked nucleoside was 2: 1.

 To extract the nucleoside product from the reaction mixture, 25 ml of ethyl acetate, 1 ml of water, 1 ml of 1N were added. hydrochloric acid and 2 ml of a saturated aqueous solution of sodium chloride. The organic layer was separated, washed with 5 ml of a saturated aqueous sodium bicarbonate solution, 5 ml of brine, and dried over magnesium sulfate.

 Quantitative analysis of HPLC showed a combined yield of the blocked beta and alpha nucleoside anomer of 42 percent.

Example 31
Preparation of Beta Anomer-Enriched 9- [1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl)] -2,6-dipivalamidoaminopurine with 2 equivalents potassium salt of 2,6-dipivalamidoaminopurine.

To 1 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 0.55 ml of triethylamine and 8.33 ml of dichloromethane at 23 ^° C. The mixture was cooled to -78 ^° C and reacted with 0.53 ml of trifluoromethanesulfonic anhydride in 0.50 ml of dichloromethane, forming the alpha-anomer-enriched 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate-1-trifluoromethanesulfonate in solution. Measures were taken to maintain the temperature of the reaction mixture below -65 ^o C.

1.85 g of a suspension of 2,6-dipivalamidoaminopurine was prepared in 30 ml of acetonitrile and contained anhydrous in a nitrogen atmosphere. 651 mg of potassium tert-butylate was added to this suspension, and the resulting mixture was stirred at 23 ^° C. for 15 minutes to form the potassium salt of 2,6-dipivalamidoaminopurine. A suspension of salt was added to 20 ml of dry dichloromethane, cooled to 0 ^° C and reacted with a solution of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate-1-trifluoromethanesulfonate enriched in alpha, and stirred for 1 hour and heated to 23 ^o C, forming the target blocked nucleoside, as confirmed by HPLC analysis.

 The anomalous ratio of beta to alpha blocked nucleoside was 2: 1.

 To extract the nucleoside product from the reaction mixture, 50 ml of ethyl acetate and 50 ml of 1N were added. of hydrochloric acid. The organic layer was separated and washed with 50 ml of 5% sodium bicarbonate. The organic layer was separated and washed with 50 ml of saturated aqueous sodium chloride and dried over magnesium sulfate.

Example 32
Preparation of 9- [1- (2'-deoxy-2,2-difluoro-3 ', 5'-di-O-benzoyl-D-ribofuranosyl)] -6-chloropurine enriched with beta anomer with 2 equivalents of 6- potassium salt chlorpurine.

To 1.4 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 14 ml of dichloromethane and 0.515 ml of triethylamine. This solution was stirred at 23 ^° C. for 15 minutes, cooled to -40 ^° C. and reacted with 0.621 ml of trifluoromethanesulfonic anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5- enriched with alpha-anomer dibenzoyl-1-trifluoromethanesulfonate in solution.

155 mg of a suspension of 6-chloropurine was prepared in 3 ml of acetonitrile and maintained anhydrous in a nitrogen atmosphere. 130 mg of potassium tert-butylate was added to the suspension, and the resulting mixture was stirred at 23 ^° C for 10 minutes, forming the potassium salt of 6-chloropurine. The salt suspension was cooled to 0 ^° C. and reacted with 2 ml of a solution of the alpha-anomer-enriched 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate, stirred for 1 hour and heated to 22 ^o C, forming the target blocked nucleoside, as confirmed by HPLC analysis.

 The anomalous ratio of beta to alpha blocked nucleoside was 2: 1.

 For the extracted nucleoside product from the reaction mixture was added 25 ml of ethyl acetate, 1 ml of water, 1 ml of 1 N. hydrochloric acid and 2 ml of a saturated aqueous solution of sodium chloride. The organic layer was separated, washed with 5 ml of a saturated aqueous sodium bicarbonate solution and 5 ml of brine, and dried over magnesium sulfate.

 Quantitative HPLC analysis showed a blocked nucleoside beta anomer yield of 27%.

Example 33
Preparation of Beta Anomer Enriched 9- [1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl)] - 2,6-dichloro-3- deazapurine with 2 equivalents of the potassium salt of 2,6-dichloro-3-deazapurine.

To 1.4 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 14 ml of dichloromethane and 0.515 ml of triethylamine. This solution was stirred at 23 ^° C for 15 minutes, cooled to -40 ^° C and reacted with 0.621 ml of trifluoromethanesulfonic anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5- enriched with alpha-anomer dibenzoyl-2,2-trifluoromethanesulfonate in solution.

82 mg of a suspension of 2,6-dichloro-3-deazapurin was obtained in 1.5 ml of acetonitrile and contained under anhydrous conditions in a nitrogen atmosphere. 49 mg of potassium tert-butylate was added to the suspension, and the resulting mixture was stirred at 23 ^° C. for 10 minutes to form the potassium salt of 2,6-dichloro-3-deazapurine. The salt suspension was cooled to 0 ^° C. and reacted with the alpha-anomer-enriched 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate in solution, stirred for 1 hour and heated to 20 ^o C, forming the target blocked nucleoside, as confirmed by HPLC analysis.

 The anomalous ratio of beta to alpha blocked nucleoside was 2.5: 1.

 To extract the nucleoside product from the reaction mixture, 25 ml of ethyl acetate, 1 ml of water, 1 ml of 1N were added. hydrochloric acid and 2 ml of a saturated aqueous solution of sodium chloride. The organic layer was separated, washed with 5 ml of a saturated aqueous sodium bicarbonate solution and 5 ml of brine, and dried over magnesium sulfate.

Quantitative analysis of HPLC showed a combined yield of blocked beta-anomeric nucleoside 21 percent, so pl. 127-129 ^o C.

Example 34
Preparation of Beta-Anomer-Enriched 9- [1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl)] -2,6-dichloropurine with 2 equivalents potassium salt of 2,6-dichloropurine.

To 1.4 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 14 ml of dichloromethane and 0.515 ml of triethylamine. This solution was stirred at 23 ^° C for 15 minutes, then cooled to -40 ^° C and reacted with 0.621 ml of trifluoromethanesulfonic anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-enriched alpha -dibenzoyl-1-trifluoromethanesulfonate in solution.

A suspension of 220 mg of 2,6-dichloropurin in 3 ml of acetonitrile was prepared and contained anhydrous in a nitrogen atmosphere. 130 mg of potassium tert-butylate was added, and the resulting mixture was stirred at 23 ^° C. for 10 minutes to form the potassium salt of 2,6-dichloropurine. The salt suspension was cooled to 0 ^° C. and reacted with the alpha-anomer-enriched 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate solution, stirred for 1 hour and heated to 22 ^° C, forming the target blocked nucleoside, as confirmed by HPLC analysis. The anomalous ratio of beta to alpha blocked nucleoside was 2.5: 1.

 To extract the nucleoside product from the reaction mixture, 25 ml of ethyl acetate, 1 ml of water, 1 ml of 1N were added. hydrochloric acid and 2 ml of a saturated aqueous solution of sodium chloride. The organic layer was separated, washed with 5 ml of a saturated aqueous sodium bicarbonate solution, 5 ml of brine, and dried over magnesium sulfate.

 Quantitative HPLC analysis showed a blocked beta-anomeric nucleoside yield of 22 percent.

Example 35
Preparation of beta-anomer-enriched 1- [1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl)] -3-carboethoxy-1,2, 4-triazole with 2 equivalents of the potassium salt of 3-carboethoxy-1,2,4-triazole.

To 1.4 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 14 ml of dichloromethane and 0.515 ml of triethylamine. This solution was stirred at 23 ^° C. for 15 minutes, cooled to -40 ^° C. and reacted with 0.621 ml of trifluoromethanesulfonic anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5- enriched with alpha-anomer dibenzoyl-1-trifluoromethanesulfonate in solution.

A suspension of 164 mg of a triazole ester in 3 ml of acetonitrile was prepared and maintained anhydrous in a nitrogen atmosphere. 131 mg of potassium tert-butylate was added, and the resulting mixture was stirred at 23 ^° C. for 10 minutes to form the potassium salt of 3-carboethoxy-1,2,4-triazole. The salt suspension was cooled to 0 ^° C and reacted with 2 ml of a solution of the alpha-anomer-enriched 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate, stirred for 40 minutes and heated to 15 ^o C, forming the target blocked nucleoside, as confirmed by HPLC analysis.

 The anomalous ratio of beta to alpha blocked nucleoside was 2.5: 1.

 To extract the nucleoside product from the reaction mixture, 25 ml of ethyl acetate, 1 ml of water, 1 ml of 1N were added. hydrochloric acid and 2 ml of a saturated aqueous solution of sodium chloride. The organic layer was separated, washed with 5 ml of a saturated aqueous sodium bicarbonate solution, 5 ml of brine, and dried over magnesium sulfate.

 Quantitative HPLC analysis showed a 14 percent yield of the blocked nucleoside beta anomer.

Example 36
Preparation of 9- [1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-riofuranosyl)] -2-amino-6-chloropurine enriched with beta anomer 2 equivalents of potassium salt of 2-amino-6-chloropurine.

To 1.4 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 14 ml of dichloromethane and 0.515 ml of triethylamine. This solution was stirred at 23 ^° C. for 15 minutes, cooled to -40 ^° C. and reacted with 0.621 ml of trifluoromethanesulfonic anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5- enriched with alpha-anomer dibenzoyl-1-trifluoromethanesulfonate in solution.

A suspension of 197 mg of 2-amino-6-chloropurine in 3 ml of acetonitrile was prepared and maintained anhydrous in a nitrogen atmosphere. 130 mg of potassium tert-butylate was added, and the resulting mixture was stirred at 23 ^° C. for 10 minutes, forming a potassium salt of 2-amino-6-chloropurine.

The salt suspension was cooled to 0 ^° C. and reacted with 2 ml of a solution enriched in the alpha-anomer of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoal-1-trifluoromethanesulfonate, stirred for 1 hour and heated to 22 ^o C, forming the target blocked nucleoside, as confirmed by HPLC analysis.

 The anomalous ratio of beta to alpha blocked nucleoside was 2: 1.

 To extract the nucleoside product from the reaction mixture, 100 ml of ethyl acetate and 10 ml of water were added and a precipitate formed. The precipitate was filtered off, washed with 5 ml of a saturated aqueous sodium bicarbonate solution, 5 ml of brine, and dried over magnesium sulfate.

 Quantitative HPLC analysis showed a 14 percent yield of the blocked nucleoside beta anomer.

Example 37
Preparation of Beta Anomer Enriched 9- [1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzene-D-ribofuranosyl)] -2,6-dipivalamidoaminopurine with 2 equivalents potassium salt of 2,6-dipivalamidoaminopurine.

To 3.78 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 30 ml of dichloromethane and 1.39 ml of triethylamine. This solution was stirred at 23 ^° C for 15 minutes, cooled to -40 ^° C and reacted with 1.68 ml of trifluoromethanesulfonic anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5- enriched with alpha-anomer dibenzoyl-1-trifluoromethanesulfonate in solution.

A suspension of 6.99 g of 2,6-dipivalamidoaminopurine was prepared in 100 ml of acetonitrile and maintained anhydrous in a nitrogen atmosphere. 2.46 g of potassium tert-butylate was added, and the resulting mixture was stirred at 23 ^° C. for 10 minutes and dried to constant weight in vacuo at 40 ^° C. to form the potassium salt of 2,6-dipivalamidoaminopurine. A salt suspension was added to 100 ml of dichloromethane, cooled to 0 ^° C. and reacted with a solution of enriched alpha-anomer 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate, stirred for 1 hours and heated to 22 ^o C, forming the target blocked nucleoside, which was confirmed by HPLC analysis.

 To extract the nucleoside product from the reaction mixture, 500 ml of ethyl acetate, 20 ml of ice, 20 ml of 1N were added. hydrochloric acid and 35 ml of a saturated aqueous solution of sodium chloride. The organic layer was separated, washed with 25 ml of a saturated aqueous sodium bicarbonate solution, 25 ml of brine, and dried over magnesium sulfate. The anomalous ratio of beta to alpha blocked nucleoside was 1.8: 1.

Quantitative HPLC analysis showed a 28 percent yield of the blocked beta-anomer of the nucleoside, so pl. 138-239 ^o C.

Example 38
Preparation of Beta-Anomer-Enriched 9- [1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl)] - 6-pivalamidoaminopurine with 2 equivalents of potassium salt 6-pivalamido aminopurine.

To 1.4 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 14 ml of dichloromethane and 0.515 ml of triethylamine. This solution was stirred at 23 ^° C. for 15 minutes, cooled to -40 ^° C. and reacted with 0.621 ml of trifluoromethanesulfonic anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5- enriched with alpha-anomer dibenzoyl-1-trifluoromethanesulfonate in solution.

A suspension of 255 mg of 6-pivalamidoaminopurine in 3 ml of acetonitrile was prepared and maintained anhydrous in a nitrogen atmosphere. 131 mg of potassium tert-butylate was added, and the resulting mixture was stirred at 23 ^° C. for 10 minutes, forming the potassium salt of 6-pivalamido aminopurine. The salt suspension was cooled to 0 ^° C. and reacted with a solution of the alpha-anomer-enriched 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate, stirred for 1 hour and heated to 22 ^° C, forming the final blocked nucleodysis, as confirmed by HPLC analysis.

 The anomalous ratio of beta to alpha blocked nucleoside was 2: 1.

 To extract the nucleoside product from the reaction mixture, 25 ml of ethyl acetate, 1 ml of water, 1 ml of 1N were added. hydrochloric acid and 2 ml of a saturated aqueous solution of sodium chloride. The organic layer was separated, washed with 5 ml of a saturated aqueous sodium bicarbonate solution, 5 ml of brine, and dried over magnesium sulfate.

 Quantitative analysis of HPLC showed a combined yield of the blocked beta and alpha nucleoside anomer of 28 percent.

Example 39
Preparation of Beta Anomer-Enriched 9- [1- (2'-deoxy-2 ', 2'-difluoro-3'-, 5'-di-O-benzoyl-D-ribofuranosyl)] - 8-bromo-7-cyano -deaza-6-pivalamidopurine with 2 equivalents of the potassium salt of 8-bromo-7-cyano-7-dease-6-pivalamidopurine.

To 1.4 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-bibenzoate was added 14 ml of dichloromethane and 0.515 ml of triethylamine. This solution was stirred at 23 ^° C. for 15 minutes, cooled to -40 ^° C. and reacted with 0.621 ml of trifluoromethanesulfonic anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl enriched with alpha-anomer -1-trifluoromethanesulfonate in solution.

A suspension of 187 ml of 8-bromo-7-cyano-7-dease-6-pivalamino-dopurin in 3 ml of acetonitrile was prepared and maintained anhydrous in a nitrogen atmosphere. 65 mg of potassium tert-butylate was added, and the resulting mixture was stirred at 23 ^° C. for 10 minutes to form the potassium salt of 8-bromo-7-cyano-7-dease-6-pivaloylamidopurine. The salt suspension was cooled to 0 ^° C. and reacted with 1 ml of a solution enriched with the alpha-anomer of 2-deoxy-2,2-difluoro-D-ribofuranosyl-1-trifluoromethanesulfonate, stirred for 1 hour and heated to 20 ^° C. target blocked nucleoside as confirmed by HPLC analysis.

 The anomalous ratio of beta to alpha blocked nucleoside was 2: 1.

 To extract the nucleoside product from the reaction mixture, 25 ml of ethyl acetate, 1 ml of water, 1 ml of 2 N were added. hydrochloric acid and 2 ml of a saturated aqueous solution of sodium chloride. The organic layer was separated, washed with 5 ml of a saturated aqueous sodium bicarbonate solution, 5 ml of brine, and dried over magnesium sulfate.

 Quantitative HPLC analysis showed a combined yield of the blocked beta and alpha nucleoside anomer of 24 percent.

Example 40
Preparation of Beta Anomer-Enriched 9- [1- (2'-deoxy-2 ','2'-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)] -2,6-dipivalamidoaminopurine with 2 equivalents of the potassium salt of 2,6-dipivalamidoaminopurine in various reaction solvents.

To 1 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 10 ml of dichloromethane and 0.36 ml of triethylamine. This solution was stirred at 23 ^° C for 15 minutes, cooled to -40 ^° C and reacted with 0.45 ml of trifluoromethanesulfonic anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5- enriched with alpha-anomer dibenzoyl-1-trifluoromethanesulfonate in solution.

A suspension of 1.85 g of 2,6-dipivalamidoaminopurine in 30 ml of acetonitrile was prepared and maintained anhydrous in a nitrogen atmosphere. 0.65 g of potassium tert-butylate was added, and the resulting mixture was stirred at 25 ^° C. for 10 minutes, forming the potassium salt of 2,6-dipivalamidoaminopurine. The salt suspension was dried in vacuum at 40 ^o C, forming a white solid with constant weight. 207 mg of purine salt was suspended in 1.5 solvent, shown in experiments AF in table. 3, presented at the end of the description, in a nitrogen atmosphere at 0 ^o C and was subjected to reaction with 1 ml of a solution enriched with alpha-anomer 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate, mixture was mixed for 1 hour and heated to 0 ^o C, forming the target blocked nucleoside, which was confirmed by HPLC analysis.

 The anomeric ratios of beta to alpha blocked nucleoside are shown below.

 To extract the nucleoside product from the reaction mixture, 25 ml of ethyl acetate, 1 ml of water, 1 ml of 1N were added. hydrochloric acid and 2 ml of a saturated aqueous solution of sodium chloride. The organic layer was separated, washed with 2 ml of a saturated aqueous sodium bicarbonate solution, 5 ml of brine, and dried over magnesium sulfate.

 Quantitative analysis of HPLC showed the yield of the blocked beta-anomer of the nucleoside shown in table. 3 (see the end of the description).

Example 41
Preparation of Beta Anomer-Enriched 9- [1- (2'-deoxy-2 ','2'-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)] -2-acetamido-6-diphenylcarbamoyloxypurine with 2 equivalents of the potassium salt of 2-acetamido-6-diphenylcarbamoyloxypurine.

To 1.4 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 14 ml of dichloromethane and 0.515 ml of triethylamine. This solution was stirred at 23 ^° C for 15 minutes, cooled to -40 ^° C and reacted with 0.621 ml of trifluoromethanesulfonic anhydride to form 2,2-difluoro-2-deoxy-D-ribofuranosyl-3,5- enriched with alpha-anomer dibenzoyl-1-trifluoromethanesulfonate in solution.

A solution of 2.56 g of 2-acetamido-6-diphenylcarbamoyloxypurine in 50 ml of hot dimethylformamide was prepared and maintained anhydrous in a nitrogen atmosphere. The solution was cooled to 25 ^° C. and 0.74 g of potassium tert-butylate was added. The resulting mixture was stirred at 23 ^° C. for 10 minutes and evaporated to an oil, which was triturated with ether, collected on a filter and dried in vacuo at 40 ^° C. to give the potassium salt of 2-acetamido-6-diphenylcarbamoyloxypurine. 496 mg of purine salt was suspended in 3 ml of dichloromethane, the suspension was cooled to 5 ^° C and reacted with a solution of the enriched alpha-anomer of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate, mixed for 1 hour and heated to 25 ^o C, forming the target blocked nucleoside, as confirmed by HPLC analysis.

 The anomeric ratio of beta to alpha blocked nucleoside was 1.8: 1.

 To extract the nucleoside product from the reaction mixture, 25 ml of ethyl acetate, 1 ml of water, 1 ml of 1N were added. hydrochloric acid and 2 ml of a saturated aqueous solution of sodium chloride. The organic layer was separated, washed with 5 ml of a saturated aqueous sodium bicarbonate solution, 5 ml of brine, and dried over magnesium sulfate. The yield of the blocked beta anomer of the nucleoside was 5.8%.

Example 42
Preparation of Beta Anomer-Enriched 9- [1- (2'-deoxy-2 ','2'-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)] -2,6-dipivalamidoaminopurine with 7 equivalents of the 2,6-potassium salt of 2,6-dipivalamidoaminopurine.

To 100 mg of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate was added 3 ml of dichloromethane and 0.036 ml of triethylamine. This solution was stirred at 23 ^° C for 15 minutes, cooled to -78 ^° C and reacted with 0.045 ml of trifluoromethanesulfonic anhydride to form 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5- enriched with alpha-anomer dibenzoyl-1-trifluoromethanesulfonate in solution.

A suspension of 1.85 g of 2,6-dipivalamidoaminopurine in 30 ml of acetonitrile was prepared and maintained anhydrous in a nitrogen atmosphere. 0.65 g of potassium tert-butylate was added, and the resulting mixture was stirred at 23 ^° C. for 10 minutes and dried in vacuo at 40 ^° C. to form the potassium salt of 2,6-dipivalamidoaminopurine, which was cooled to -78 ^° C. The purine salt was reacted with a solution of enriched alpha-anomer 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate at 23 ^o C, was mixed for 1.5 hours and heated to 22 ^o C, forming the target blocked nucleoside as confirmed by HPLC analysis data.

 To extract the nucleoside product from the reaction mixture, 25 ml of ethyl acetate, 1 ml of ice, 1 ml of 1N were added. hydrochloric acid and 2 ml of a saturated aqueous solution of sodium chloride.

 The organic layer was separated, washed with 5 ml of a saturated aqueous sodium bicarbonate solution, 5 ml of brine, and dried over magnesium sulfate.

 The anomalous ratio of beta to alpha blocked nucleoside was 2.7: 1.

Example 43
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 3 equivalents of bis- trimethylsilylcytosine.

Bis-trimethylsilicytosine was prepared by combining 292 mg of cytosine with 2 ml of hexamethyldisilazane, 11 mg of ammonium sulfate and 5 ml of xylene and heating the solution under reflux for one hour to form a homogeneous solution. Excess xylenes and hexamethyldisilazane was removed, leaving a molten residue of bis-trimethylsilylcytosine. 400 mg of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate dissolved in 2 ml of xylenes was added to the molten bis-trimethylsilylcytosine, and the xylenes were removed. The temperature of the reaction mixture was maintained at 160 ^° C. for 15 minutes.

 HPLC analysis confirmed the completion of the reaction. The anomalous ratio of beta to alpha blocked nucleoside was 1: 1.3.

 To extract the nucleoside product, the reaction mixture was cooled, diluted with 50 ml of ethyl acetate and washed with 50 ml of 1 N. of hydrochloric acid.

Example 44
Preparation of 9- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-ketopyrimidin-2-one enriched with beta anomer with 3 equivalents of bis- trimethylsilyluracil.

Bis-trimethylsilyl uracil was prepared by combining 295 mg of uracil with 5 ml of hexamethyldisilazane, 11 mg of ammonium sulfate and 10 ml of 1,2-dichloroethane. The solution was heated to 110 ^° C. for one hour to form a homogeneous solution, and the excess of xylenes and hexamethyldisilazane was removed to give molten bis-trimethylsilyluracil.

200 mg of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate was added to the molten bis-trimethylsilyluracil. The temperature of the reaction mixture was maintained at 150 ^° C. for 2 hours.

 HPLC analysis confirmed the completion of the reaction. The anomalous ratio of alpha to beta blocked nucleoside was 1: 1.8.

 To extract the nucleoside product, the reaction mixture was cooled, diluted with 50 ml of ethyl acetate and washed with 50 ml of 1 N. of hydrochloric acid.

Example 45
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 10 equivalents of bis- trimethylsilylcytosine.

To 1.12 g of molten bis-trimethylsilicytosine was added 200 mg of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate. The temperature of the reaction mixture was maintained at 130 ^° C. for 1 hour.

 HPLC analysis confirmed the completion of the reaction. The anomalous ratio of the blocked nucleoside beta product to alpha blocked nucleoside was 1.7: 1.

 To extract the nucleoside product, the reaction mixture was diluted with 100 ml of ethyl acetate and washed with 100 ml of 1 N. of hydrochloric acid. Quantitative analysis of the HPLC of the organic layer showed that the yield of the blocked beta nucleoside anomer was 50%.

Example 46
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-acetamidopyrimidin-2-one enriched with beta anomer with 3 equivalents of bis- trimethylsilyl-N-acetylcytosine.

To 500 ml of bis-trimethylsilyl-N-acetylcytosine was added 980 mg of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate. The temperature of the reaction mixture was maintained at 100 ^° C. for 3 hours.

 HPLC analysis confirmed the completion of the reaction. The anomalous ratio of beta to alpha blocked nucleoside was 1.4: 1.

 To extract the nucleoside product, the reaction mixture was cooled, diluted with 25 ml of ethyl acetate and washed with 25 ml of 1 N. of hydrochloric acid. The aqueous layer was washed with 30 ml of ethyl acetate. Quantitative analysis of the HPLC of the organic layer showed that the yield of the blocked nucleoside beta anomer was 34 percent.

Example 47
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-acetamidopyridin-2-one enriched with beta anomer with 3 equivalents of bis- trimethylsilyl-N-acetylcytosine.

To 393 ml of bis-trimethylsilyl-N-acetylcytosine, 200 mg of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-methanesulfonate was added. The temperature of the reaction mixture was maintained at 100 ^° C. for 1 hour.

 The anomalous ratio of beta to alpha blocked nucleoside product was 2.3: 1.

 To extract the nucleoside product, the reaction mixture was diluted with 40 ml of ethyl acetate and washed with 25 ml of 1 N. of hydrochloric acid. Quantitative analysis of the HPLC of the organic layer showed that the yield of the nucleoside beta anomer was 27 percent.

Example 48
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-aminopyrimidin-2-one enriched with beta anomer with 20 equivalents of bis- trimethylsilylcytosine.

Bis-trimethylsilylcytosine was obtained by combining 4.9 g of cytosine with 90 ml of hexamethyldisilazane, 581 mg of ammonium sulfate and 2 ml of xylene and heating the solution for two hours to form a homogeneous solution. Excess hexamethyldisilazane was removed and a white residue formed. To a solution of bis-trimethylsilylcytosine was added 1 g of 2-deoxy-2,2-difluoro-D-ribofuranoside-3,5-dibenzoyl-1-α-methanosulfonate dissolved in 5 ml of acetonitrile, and acetonitrile was removed. The temperature of the reaction mixture was maintained at 130 ^° C. under vacuum for 1 hour.

 HPLC analysis showed completion of the reaction. The anomalous ratio of beta to alpha blocked nucleoside product was 3.9: 1.

 To extract the nucleoside product, the reaction mixture was diluted with 100 ml of dichloromethane and washed successively with 100 ml of 1 N. hydrochloric acid and 200 ml of 5% sodium bicarbonate, and then 200 ml of saturated sodium chloride. The organic layer was dried over magnesium sulfate, filtered and evaporated to 1.03 g of a yellow solid.

 Quantitative analysis of HPLC showed that the yield of the beta-anomer of the nucleoside was 43 percent.

 In the table. 4 (see end of description) shows how the selected carbohydrate, reaction temperature and molar equivalent of a nucleoside affect the yield and anomeric ratio of the nucleoside product.

 (N / D) means not determined. Carbohydrates (carbo.) Are hydroxy protected. The designations α or β-OMs are alpha or beta-2,2-difluoro-2-deoxy-D-ribofuranosyl-3,5-dibenzoyl-1-methanesulfonate and β- or α-OTs are beta or alpha-2, 2-difluoro-2-deoxy-D-ribofuranosyl-3,5-dibenzoyl-1-toluenesulfonate. The yield data are presented based on the total amount of carbohydrate and calculated using quantitative HPLC analysis in the reverse phase, in which the peak of the corresponding product solution was compared with the standard, 1- (2'-deoxy-2 ', 2'-difluoro-3', 5 '-di-O-benzoyl-beta-D-ribofuranosyl) -4-aminopyrimidin-2-one. The protective group of the nucleoside base in each example is trimethylsilyl.

Example 49
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4-pivalamidopyrimid-2-one enriched in beta anomer in acetonitrile.

N-pivalamidocytosine (1.0 g, 5.5 mmol) was suspended in acetonitrile (15.0 ml) and treated with potassium tert-butylate (0.062 g, 5.5 mmol) and stirred in a nitrogen atmosphere at 25 ^° C, forming the potassium salt of N-pivaloylcytosine.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α- (p-bromobenzenesulfonate (2.99 g, 5.0 mmol) in acetonitrile (10.0 ml) was added to the above salt, and the whole mixture underwent a reaction for 5.5 hours at 65 ^o C, forming a blocked nucleoside.

 HPLC analysis confirmed the completion of the reaction and showed an anomeric ratio of beta to alpha 3.9: 1.

To isolate the nucleoside product, the reaction mixture was partitioned between ethyl acetate and water, and the organic layer was washed with sodium bicarbonate and dried over magnesium sulfate. Column chromatography (silica gel, 6: 4 ethyl acetate toluene) afforded 0.700 g of the expected product in 20% yield, mp. 191 - 193 ^o C.

Example 50
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4- (N-pivalamido) aminopyrimide-2-one enriched with beta-anomer in acetonitrile.

N-pivalamidocytosine (0.098 g, 0.5 mmol) was suspended in acetonitrile (1.5 ml) and treated with potassium tert-butylate (0.062 g, 0.55 mmol) and stirred in a nitrogen atmosphere at 25 ^° C, giving the potassium salt of N-pivaloylcytosine.

To the above salt was added 2-deoxy-2,2-difluoro-D-ribofuranosoyl-3,5-dibenzoyl-1-α-iodide (0.244 g, 0.5 mmol) in acetonitrile (1.5 ml), and the whole mixture was reacted for 24 hours at 60 ^o C, giving a blocked nucleiside.

 HPLC analysis confirmed the completion of the reaction and showed an anomeric ratio of beta to alpha 1.13: 1.

Example 51
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -1,2,4-triazole-3-carbonitrile a enriched with beta anomer cetonitrile.

1,2,4-triazole-3-carbonitrile (0.101 g, 1.03 mmol) was suspended in acetonitrile (10 ml and treated with sodium hydride) (0.445 g, 1.12 mmol) and stirred in a nitrogen atmosphere at 25 ^o C to form the corresponding sodium salt triazole. To the above salt was added 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-bromide (0.451 g, 1.02 mmol) in acetonitrile (10 ml), and the whole mixture was reacted in for 78 hours at 82 ^o C, forming a blocked nucleoside.

 HPLC analysis confirmed the completion of the reaction and showed an anomeric ratio of beta to alpha 1.2: 1.

To isolate the nucleoside product, the reaction mixture was evaporated, forming an oily solid, diluted with ethyl acetate, washed with sodium bicarbonate and dried over magnesium sulfate and concentrated. The residue crystallized from the standard, giving 30 mg of the target product with a yield of 6%, so pl. 225 - 226 ^o C. MC (FD) M / Z 455 (M + I).

Elemental analysis for C ₂₂ H ₁₆ F ₂ N ₄ O ₅ : (Theoretical) C 58.15, H 3.55, N 12.33. (Empirical) C 58.36, H 3.79, N 12.10.

Example 52
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -1,2,4-triazole-3-carbonitrile enriched beta-anomer acetonitrile.

1,2,4-triazole-3-carbonitrile (0.272 g, 2.89 mmol) was suspended in acetonitrile 20 ml), treated with sodium hydride (0.094 g, 2.7 mmol) and stirred in a nitrogen atmosphere at 25 ^o C, forming the triazole sodium salt.

To this salt was added 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-iodide (0.941 g, 1.9 mmol) in acetonitrile (20 ml), and the whole mixture was reacted for 48 hours at 82 ^o C, forming a blocked nucleoside.

 HPLC analysis confirmed the completion of the reaction and showed an anomeric ratio of beta to alpha 3.5: 1.

To isolate the nucleoside product, the reaction mixture was evaporated, forming an oily solid, diluted with ethyl acetate, washed with sodium bicarbonate, dried over magnesium sulfate and concentrated. The residue crystallized from ethanol, giving 0.421 g of the target product; so pl. 225-226 ^o C with a yield of 48%.

 MC / FD / M / Z455 / M + 1 /.

Elemental analysis for C ₂₂ H ₁₆ F ₂ N ₄ O ₅ : (Theoretical) C 58.15. H 3.55, N 12.33. (Empirical) C 58.35, H 3.65, N 12.33.

Example 53
Obtaining enriched (9) regioisomer-beta anomer 1- (2'-deoxy-2'2, 2'-difluoro-3 ', 5'-di-O-benzoyl-D-ribofuranosyl) -6-cyanopurine in N, N-dimethylacetamide.

6-cyanopurin (0.92 g, 6.35 mmol) was suspended in N, N-dimethylacetamide (12 ml) and treated with sodium hydrate (0.396 g, 8.25 mmol) and stirred under a nitrogen atmosphere at 25 ^° C, giving the 6-cyanopurin sodium salt.

To the above salt was added 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-iodide (3.09 g, 6.35 ml) in N, N-dimethylacetamide (4 ml), the whole mixture was reacted for 5 hours at 70 ^° C., forming a blocked nucleoside.

 HPLC analysis confirmed the completion of the reaction and showed an anomeric ratio of beta to alpha 1.2: 1.

 HPLC analysis confirmed the completion of the reaction and showed an anomeric ratio of beta to alpha 1.2: 1.

To isolate the nucleoside product, the reaction mixture was cooled, the solvent was removed in vacuo, the residue was dissolved in ethyl acetate, washed with a 0.2 M solution of lithium chloride, dried over magnesium sulfate, and concentrated. Column chromatography (silica gel, toluene / ethyl acetate 9: 1) gave 0.21 g of the expected product in 6.5% MC (FD) 506 (M + 1) yield. Elemental analysis for C ₂₅ H ₁₇ F ₂ N ₅ O ₅ : (Theoretical) C 59.41, H 3.39, N 13.86. (Empirical) C 59.85, H 3.49, N 13.48.

Example 54
Obtaining enriched with (9) regioisomer-beta anomer 1- (2'-deoxyoxy-2, '2' -difluoro-3 ', 5'-di-O-benzoyl-D-rifuranosyl) -2,6- (dipivalamido) diaminopurine in N, N-dimethylacetamide.

2,6- (dipivalamido / diaminopurin) 0.159 g 0.5 mmol) was suspended in N, N-dimethylacetamide (1.0 ml) and treated with potassium tert-butylate (0.062 g, 0.55 mmol) and stirred in a nitrogen atmosphere at 25 ^o C, forming potassium salt of 2,6- (dipivaloid) diaminopurine.

To the above salt was added 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α- (p-bromobenzene) sulfonate (0.299 g, 0.5 mmol) in N, N-dimethylacetamide (0.5 ml), and the whole mixture was reacted for 6 hours at 60 ^° C. to form a blocked nucleoside.

 HPLC analysis confirmed the completion of the reaction and indicated an anomeric ratio of beta to alpha 1.9: 1 of the target product.

 To isolate the nucleoside product, the reaction mixture was cooled, and the solvent was removed in vacuo. The residue was diluted with ethyl acetate, washed with sodium bicarbonate, dried over magnesium sulfate and concentrated to an oil. Chromatography on a column (silica geltoluene / ethyl acetate 1: 1) gave 0.141 g of both alpha and beta nucleoside products with a yield of 28%. MC (FD) 679 (M + 1).

Example 55
Obtaining the enriched (9) -regioisomer-beta anomer 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -2,6 - (dipivalamido ) diaminopurine in acetonitrile
2.6- (dipivalamido / diaminopurin / 0.159 g, 0.5 mmol) was suspended in acetonitrile /1.5 ml and treated with potassium tert-butylate (0.062 g, 0.55 mmol) and stirred in a nitrogen atmosphere at 25 ^o C to form a potassium salt of 2.6 - (dipivalamido) diaminopurine.

To this salt was added 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-iodide (0.244 g, 0.5 mmol) in acetonitrile (1.5 ml), and the whole mixture was reacted in for 16 hours at 60 ^o C, forming a blocked nucleoside.

 HPLC analysis confirmed the completion of the reaction and indicated an anomeric ratio of beta to alpha 2.2: 1.

 To isolate the nucleoside product, the reaction mixture was diluted with ethyl acetate, the organic layer was washed with sodium bicarbonate, dried over magnesium sulfate, separated and concentrated to an oil. Column chromatography (silica gel, 1: 1 toluene / ethyl acetate) followed by recrystallization gave 0.085 g of the expected product in 25% yield. MC (FD) 679 (M + 1).

Example 56
Preparation of 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl) -4- (benzylamino) pyrimid-2-one enriched with beta anomer , N-dimethylacetamide.

N-benzyolcytosine (0.099 g, 0.493 mmol) was suspended in N, N-dimethylacetamide (2.0 ml) and treated with sodium hydride (0.0256 g, 0.534 mmol) was mixed under nitrogen at 25 ^° C to form the sodium salt of N-benzylcytosine.

To this salt was added 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-iodide (0.201 g, 0.411 mmol) in N, N-dimethylacetamide (1.5 ml), and the whole mixture was subjected to reaction for 5 hours at 23 ^o C with the formation of a blocked nucleoside.

 HPLC analysis confirmed the completion of the reaction and indicated an anomeric ratio of beta to alpha 1.9: 1.

 The reaction solvents were removed in vacuo and the residue was dissolved in ethyl acetate, washed with sodium bicarbonate, dried over magnesium sulfate and concentrated to an oil. Column chromatography (silica gel, 9: 1 toluene / ethyl acetate) afforded 0.015 mg of the expected product in 6.5% yield. MC (FD) 562 (M + 2).

Example 57
Preparation of Ethyl 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-D-ribofuranosyl-1,2,4-triazole-3-carbosilate-enriched beta-anomer) in N, N-dimethylacetamide.

Ethyl 1,2,4-triazole-3-carboxylate (0.723 g, 5.13 mmol) was suspended in N, N-dimethylacetamide (2.5 ml), treated with sodium hydride (0.123 g, 5.13 mmol) and stirred in a nitrogen atmosphere at 25 ^o C with the formation of the sodium salt of triazole.

To this salt was added 2-deoxy-2,22-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-iodide (2.0 g, 4.11 mmol) in N, N-dimethylacetamide (2.5 ml), and the whole mixture was subjected to reaction for 24 hours at 23 ^o C, forming a blocked nucleoside. HPLC analysis confirmed the completion of the reaction and showed an anomeric ratio of beta to alpha 3: 1.

 The crude reaction mixture was purified by removal of the solvent under reduced pressure and column chromatography (silica gel, toluene / ethyl acetate 9: 1). The combined theoretical yield of alpha and beta regioisomers (A and B below) of blocked nucleosides was 67 percent.

 A.

 Ethyl 1- (2'-deoxy-2,2'-difluoro-3 ', 5'-di-O-benzoyl-β-D-ribofuranosyl) -1,2,4-triazole-3-carboxylate (436 mg, 21.2% yield).

Перекристаллизация "A" из смеси этилацетат : изооктан давала 267 мг чистого бета-аномера с выходом 13%.

Recrystallization of “A” from ethyl acetate: isooctane gave 267 mg of pure beta anomer in 13% yield.

 B.

 Ethyl 1- (2'-deoxy-2 ', 2'-difluoro-3', 5'-di-O-benzoyl-β-D-ribofuranosyl) -1,2,4-triazole-5-carboxylate (855 mg , 41.5% yield).

Пример 58
Получение обогащенного бета-аномером 2-дезокси-2,2-дифтор-D-рибофуранозил-1 -β- (2-амино-6-хлорпурина) в диметилацетамиде.

Example 58
Preparation of 2-deoxy-2,2-difluoro-D-ribofuranosyl-1-β- (2-amino-6-chloropurine) enriched beta anomer in dimethylacetamide.

To a suspension of 2-amino-6-chloropurine (82.6 mmol, 14.0 g) in dimethyl acetamide (900 ml) at 0 ^° C in a nitrogen atmosphere was added powdered potassium hydroxide (99.12 mmol, 5.5 g). The mixture was stirred for 30 minutes to form a solution. 2-Deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-α-iodide (82.6 mmol, 40.31 g) in dimethylacetamide (450 ml) was added. The reaction mixture was allowed to warm to room temperature and was stirred under nitrogen overnight.

 The product was extracted by addition of ethyl acetate and brine. The organic layer was washed sequentially with 1 N. HCl, saturated sodium bicarbonate, water and saline. The organic layer was then dried over sodium sulfate and evaporated in vacuo.

 The crude product was purified by silica gel chromatography to give a 3: 1 anomeric ratio of beta to alpha 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1- (2-amino-6-chloropurine).

¹ H NMR (300 MHz, CD ₃ CD), δ 4.68 (m, 2H), 4'-H, 5'-aH), 4.90 (m, 1H, 5 'bH), 6.02 (m, 1H , 3'-H), 6.29 (m, 1H, 1'-H), 7.53 (m, 6H, Bz), 7.92 (s, 1H, 8'-H), 8.05 (m, 4H, Bz).

The dibenzoyl intermediate (0.49 mmol, 260 mg) was released by suspending it in methanol at 0 ^° C and saturating the mixture with anhydrous ammonia. The resulting solution was warmed to room temperature and stirred overnight. The solution was then purged with nitrogen and evaporated. The target product was then purified by washing with a non-polar solvent, such as methylene chloride, to remove benzoate products. The beta anomer was separated by reverse phase HPLC.

¹ H NMR (300 MHz, CD ₃ CD), δ 3.90 (m, 3H, 4'-H, 5'-H), 4.58 (m, 1H, 3'-H), 6.27 (dd. , 1H, 1'-H), 8.31 s, 1H, 8-H).

Getting 1
Enriched with alpha-anomer 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-di-O-benzoyl-1-methanosulfonate.

Triethylamine (0.025 ml) was added to a solution of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate (40 mg) in methylene chloride (0.5 mg). After stirring at room temperature for 30 minutes, the whole mixture was cooled to -78 ^° C, then methanesulfonyl chloride (0.01 ml) was added. The reaction temperature was maintained between -78 ^° C and -80 ^° C for 30 minutes, then warmed to room temperature.

HPLC analysis showed that the reaction was completed. The anomeric ratio of the target compound alpha to beta according to the determination using ¹⁹ F NMR analysis: was 4: 1.

Getting 2
Alpha anomer of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-di-O-benzoyl-1-methanesulfonate.

To a solution of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoate (60 g, 95% purity) in dichloromethane (600 ml) was added triethylamine (31.5 ml, 1.5 equivalents). After stirring at room temperature for 30 minutes, the mixture was cooled to -78 ^° C. After 5 minutes, methanesulfonyl chloride (14 ml, 1.2 equiv.) In dichloromethane (140 ml) was added to the mixture. The reaction temperature was maintained between -78 ^o C and -80 ^o C in a nitrogen atmosphere for one hour.

 HPLC analysis showed that the reaction was complete. The homogeneous ratio of the target compound according to HPLC analysis was 3.53: 1 alpha to beta.

To isolate the target compound, the reaction mixture was washed with water, 1 N. HCl solution and 5% sodium bicarbonate solution (30 ml each). The organic layer was separated and dried over anhydrous magnesium sulfate. The target compound (31.5 g) was obtained with a yield of 46 percent, so pl. 88-89 ^o C; [α] _D (c 1.01, CHCl ₃ ) + 84.2; [α] _{365 am} ⁺ 302.0 ^o . Elemental analysis: C ₂₀ H ₁₈ O ₈ SF ₂ :
Calculated: C 52.63, H 3.98, F 8.33, S 7.02 (456.4).

Found: C, 52.92; H, 3.82; F, 8.33; S, 7.30;
¹ H NMR (CDCl ₃ ), δ = 3.17 (CH ₃ ), 4.66 and 4.76 (C-5H), 4.84 (C-4H), 5.57 (C-3H), 6.13 (C-1H), ¹³ C NMR ( CDCl ₃ ), δ = 40.22 (CH ₃ ), 62.51 (C-5H), 71.03 (C-3H), J _{C, F} = 18.3, 38.5 Hz), 82.75 (C-4H), 99.59 (C- 1H, J _{C, F} = 25.5, 48.3 Hz), 122.24 (C-2H, J _{C, F} = 259, 286 Hz).

Getting 3
Enriched with alpha-anomer 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-di-O-benzoyl-1-methanesulfonate.

To the anomeric mixture of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-O-benzoyl-1-methanesulfonate (1.0 g, 97% beta-anomer) in acetonitrile (10 ml) was added N, N dimethylbenzylammonium methanesulfonate (100 mg). The mixture was stirred and heated to reflux. HPLC analysis was used to determine the ratio of alpha to beta of the target product and showed the following:
Time / Clock - Alpha / Beta
0 - 1:32
16 - 1.0: 1.4
24 - 2.3: 1.0
Getting 4
Enriched with the alpha anomer, 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-di-O-benzoyl-1-methanesulfonate.

To the anomeric mixture of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-di-O-benzoyl-1-methanesulfonate (29.1 g, 50% beta-anomer) in dichloromethane and n-propyl acetate ... was heated up to 90 ^o C to remove dichloromethane. The mixture was cooled to 50-60 ^° C and a mixture of triethylamine (5.33 ml, 0.55 equiv.) And methanesulfonic acid (2.04 ml, 0.55 equiv.) Was added. The resulting mixture was heated to 95-97 ^o C and mixed. The mixture contained 23.2 g of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-di-O-benzoyl-1-methanosulfonate. To determine the ratio of alpha to beta of the target product, HPLC analysis was used and showed the following:
Time (hours) - Alpha / Beta
4 - 3: 1g

Claims (20)

1. The method of obtaining enriched beta anomers of nucleosides of General formula I

where T is fluorine;
R represents a nucleoside selected from the group consisting of radicals

where R is selected from the group consisting of hydrogen, alkyl, substituted alkyl and halogen;
R ₂ selected from the group consisting of hydroxy, halogen, primary amine or secondary amine;
R ₄ , R ₅ and R _{6 are} independently selected from the group consisting of hydrogen, —OH, —NH ₂ , N (alkyl) W, halogen;
Q is selected from the group consisting of CH, CR ₈ or N, where R _{8 is} selected from the group consisting of halogen or cyano,
characterized in that SN ₂ is optionally substituted in a suitable solvent of the sulfonyloxy group (Y) from the alpha-anomer-enriched carbohydrate of formula II

where X is independently selected from hydroxy protecting groups;
T has the meanings defined above
at least the molar equivalent of the nucleobase (R ″) selected from the group consisting of

where R ₁ , R ₂ , R ₄ , R ₅ , R ₆ and Q are as defined above;
Z represents a hydroxy protecting group;
W represents an amino protecting group;
M ^⊕ represents a cation,
moreover, SN ₂ substitution is carried out at a temperature of 17 ... -120 ^o With and release with the formation of the compounds of formula I. 2. The method according to claim 1, characterized in that R "is selected from the group consisting of

where R ₁ selected from the group consisting of hydrogen, alkyl, substituted alkyl and halogen;
Z represents a hydroxy protecting group;
W represents an amino protecting group;
Y is selected from the group consisting of alkylsulfonyloxy, arylsulfonyloxy, substituted alkylsulfonyloxy and substituted arylsulfonyloxy group,
moreover, the process is carried out in a solution having a carbohydrate concentration of more than 20 wt.% per unit volume of solvent, and an inert solvent with a boiling point of more than 70 ^o C. is used as a solvent. 3. The method according to claim 1, characterized in that R "is selected from the group consisting of

where R ₁ selected from the group consisting of hydrogen, alkyl, substituted alkyl and halogen;
R ₂ selected from the group consisting of hydroxy, halogen, primary amino or secondary amino;
Z represents a hydroxy protecting group;
W represents an amino protecting group;
Y is selected from the group consisting of trifluoromethanesulfonyloxy, 1,1,1-trifluoroethanesulfonyloxy, octafluorobutanesulfonyloxy and nonafluorobutanesulfonyloxy,
moreover, SN ₂ substitution is carried out at a temperature of from -120 to 25 ^o With using a low-freezing inert solvent. 4. The method according to claim 1, characterized in that R "is selected from the group consisting of

where R ₁ selected from the group consisting of hydrogen, alkyl, substituted alkyl and halogen;
Z represents a hydroxy protecting group;
W represents an amino protecting group;
Y is selected from the group consisting of alkylsulfonyloxy, arylsulfonyloxy, substituted alkylsulfonyloxy and substituted arylsulfonyloxy,
moreover, the process is carried out in the presence of a catalyst.  5. The method according to claim 4, characterized in that the catalyst is selected from highly ionized salts, which are essentially soluble in the solvent and contain a non-nucleophilic anion.  6. The method according to claim 4 or 5, characterized in that the solvent is selected from polar, non-nucleophilic solvents. 7. The method according to claim 1, characterized in that R "is selected from the group consisting of

where R ₂ selected from the group consisting of hydroxy, halogen, primary amino or secondary amino;
R ₄ , R ₅ , R _{6 are} independently selected from the group consisting of hydrogen, —OH, NH ₂ , N (alkyl) W, halogen;
Q is selected from the group consisting of CH, CR ₈ and N, where R _{8 is} selected from the group consisting of halogen or cyano;
Z represents a hydroxy protecting group;
W represents an amino protecting group;
M ^⊕ represents a cation;
Y is selected from the group consisting of trifluoromethanesulfonyloxy, 1,1,1-trifluoroethanesulfonyloxy, octafluorobutanesulfonyloxy and nonafluorobutanesulfonyloxy;
and the solvent is an inert solvent with a freezing point below 26 ^o C. 8. The method according to claim 1, characterized in that the process is carried out in the absence of a solvent and R ″ is selected from the group consisting of

where R ₁ selected from the group consisting of hydrogen, alkyl, substituted alkyl and halogen;
Z represents a hydroxy protecting group;
W represents an amino protecting group, wherein Y is selected from the group consisting of alkylsulfonyloxy, arylsulfonyloxy, substituted alkylsulfonyloxy and substituted arylsulfonyloxy. 9. The method according to claim 8, characterized in that the reaction is carried out at a temperature of from 100 to 160 ^o C. 10. The method according to claim 1, characterized in that R "is selected from the group consisting of

where R ₁ selected from the group consisting of hydrogen, alkyl, substituted alkyl and halogen;
R ₂ selected from the group consisting of hydroxy, halogen, primary amino and secondary amino;
R ₄ , R ₅ , R _{6 are} independently selected from the group consisting of hydrogen, —OH, NH ₂ , N (alkyl) W, halogen;
Q is selected from the group consisting of CH, CR ₈ and N, where R _{8 is} selected from the group consisting of halogen or cyano;
Z represents a hydroxy protecting group;
W represents an amino protecting group;
Y is selected from the group consisting of alkylsulfonyloxy, arylsulfonyloxy, substituted alkylsulfonyloxy and substituted arylsulfonyloxy.  11. The method according to any one of claims 1 to 10, characterized in that the hydroxy protecting group X of the compound of formula II is benzoyl.  12. The method according to any one of claims 1 to 10, characterized in that the sulfonyloxy group Y of the compounds of formula II is methanesulfonyloxy.  13. The method according to claim 2, characterized in that the sulfonyloxy group Y of the compounds of formula II is methanesulfonyloxy.  14. The method according to claim 4, characterized in that the sulfonyloxy group Y of the compounds of formula II is methanesulfonyloxy.  15. The method according to claim 5, characterized in that the sulfonyloxy group Y of the compounds of formula II is methanesulfonyloxy.  16. The method according to claim 6, characterized in that the sulfonyloxy group Y of the compounds of formula II is methanesulfonyloxy.  17. The method of claim 8, wherein the sulfonyloxy group Y of the compound of formula II is methanesulfonyloxy.  18. The method according to claim 9, characterized in that the sulfonyloxy group Y of the compounds of formula II is methanesulfonyloxy.  19. The method according to claim 10, characterized in that the sulfonyloxy group Y of the compounds of formula II is methanesulfonyloxy.  20. The method according to claim 1, characterized in that the sulfonyloxy group Y of the compounds of formula II is trifluoromethanesulfonyloxy.

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